CN111971052A

CN111971052A - Method for allogeneic hematopoietic stem cell transplantation

Info

Publication number: CN111971052A
Application number: CN201980024106.6A
Authority: CN
Inventors: 埃弗雷特·于尔托·迈耶; 罗伯特·S·内格林
Original assignee: Leland Stanford Junior University
Current assignee: Leland Stanford Junior University
Priority date: 2018-02-08
Filing date: 2019-02-07
Publication date: 2020-11-20
Also published as: US20210030797A1; US20220380729A1; EP3749334A2; JP2024041938A; AU2019218792A1; WO2019157158A2; US20210002613A1; EP3749334A4; US11952588B2; WO2019157158A3; US20210015862A1; US11447747B2; CA3090175A1; JP7422667B2; JP2021512903A

Abstract

The present invention provides methods for improving hematopoietic stem cell transplantation, including methods of enhancing the ability to prevent graft-versus-host disease while maintaining an effective immune response (e.g., graft-versus-tumor immune response). The present invention provides methods of administering, e.g., hematopoietic stem progenitor cells, regulatory T cells, and conventional T cells, wherein the conventional T cells are administered after administration of the hematopoietic stem progenitor cells and regulatory T cells. The invention also provides methods of administering, e.g., hematopoietic stem progenitor cells, regulatory T cells, and conventional T cells, wherein the regulatory T cells are not cryopreserved prior to administration.

Description

Method for allogeneic hematopoietic stem cell transplantation

Cross-referencing

This patent application claims priority to U.S. provisional patent application No. 62/628,105, filed on 8.2.2018, which is incorporated herein by reference in its entirety.

Background

Patients at high risk of hematologic malignancies (e.g., relapsed leukemia and lymphoma after first remission or relapsed refractory leukemia and lymphoma) are rarely cured by standard chemotherapy. Myeloablative allogeneic Hematopoietic Cell Transplantation (HCT) can increase the survival of the patient, wherein the disease-free survival of the patient is between 10% and 50% and the non-recurrent mortality is between 30% and 50%. Early morbidity and mortality associated with acute graft versus host disease (aGVHD) and long-term morbidity associated with chronic GVHD are major factors limiting HCT success.

Is incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

Disclosure of Invention

In some embodiments, the present invention discloses a method of treating a human subject in need of treatment described herein, comprising administering to the human subject: a) a population of Hematopoietic Stem Progenitor Cells (HSPCs); b) a cell population comprising regulatory T cells (tregs); and c) a population of conventional T cells (Tcon); wherein the HSPC population and the Treg-comprising cell population are administered prior to the administration of the Tcon population; and wherein said population of cells comprising tregs has not been cryopreserved prior to said administration thereof.

In some embodiments, the present invention discloses a method of treating a human subject in need of treatment described herein, comprising administering to the human subject: a) a population of Hematopoietic Stem and Progenitor Cells (HSPCs); b) a cell population comprising regulatory T cells (tregs); and c) a population of conventional T cells (Tcon); wherein the population of HSPCs and the population of cells comprising Tregs are administered prior to administration of the population of conventional T cells; and wherein said human subject has not developed Graft Versus Host Disease (GVHD) within 30 days after said administration of said Tcon population.

In some embodiments, the present invention discloses a method of treating a human subject in need of treatment described herein, comprising administering to the human subject: a) a population of Hematopoietic Stem Progenitor Cells (HSPCs); b) a cell population comprising regulatory T cells (tregs); and c) a population of conventional T cells (Tcon); wherein the HSPC population and the Treg-comprising cell population are administered prior to the administration of the Tcon population; wherein the population of cells comprising Tregs comprises CD45⁺Cells, wherein more than 90% of said CD45⁺The cells are tregs.

Drawings

Figure 1 provides patient characteristics for a clinical trial comprising administration of HSPCs and tregs prior to Tcon administration.

Figure 2 illustrates an original and modified clinical protocol diagram.

Figure 3 illustrates the enrichment of Treg cells from peripheral blood apheresis products administered to a subject.

Fig. 4 illustrates the suppressive ability of a population of cells comprising tregs in a Mixed Lymphocyte Reaction (MLR).

Figure 5 illustrates a dose escalation protocol for a clinical trial comprising administration of HSPCs and tregs prior to Tcon administration.

Fig. 6 provides a schematic of graft generation and administration of fresh tregs.

Figure 7 shows the cell dose yield of tregs from donors in clinical trials.

Figure 8 provides details of the dose of cells administered to a subject in a clinical trial.

FIG. 9 summarizes the clinical prognostic data for the clinical trial.

Figure 10 illustrates immune cell reconstitution at standardized time points after HCT.

Figure 11 illustrates Treg characteristics at day 60 post HCT and Treg TCR repertoire diversity at day 90 post HCT.

Detailed description of the preferred embodiments

The following description and examples detail embodiments of the invention. It is to be understood that the invention is not limited to the particular embodiments described herein, as variations in actual practice are possible. Those skilled in the art will recognize that numerous variations and modifications may be made to the present invention, all of which are within the scope of the present invention.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

The term "about" and its grammatical equivalents as used herein with respect to a reference value and its grammatical equivalents may include a range of values by the addition or subtraction of 10% to the value. For example, a value of "about 10" includes values from 9 to 11. The term "about" with respect to a reference value can also include a range of values that are plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the recited value.

Summary of The Invention

The present invention provides methods for improving allogeneic hematopoietic stem cell transplantation (HCT). In some embodiments, the methods disclosed herein preserve the Graft Versus Tumor (GVT) effect of HCT administration to a cancer subject while preventing or reducing the occurrence of Graft Versus Host Disease (GVHD).

HCT refers to the transplantation of multipotent Hematopoietic Stem Progenitor Cells (HSPCs), usually from donor bone marrow, peripheral blood or umbilical cord blood, into a recipient. The recipient may receive a myeloablative conditioning, which may kill hematopoietic cells, including tumor cells and host immune cells. HSPCs transplanted into recipients can then reconstitute the hematopoietic compartment. HCT is useful in the treatment of cancer because donor T cells can exert an anti-tumor effect, known as Graft Versus Tumor (GVT). HCT can increase survival for patients with refractory hematological malignancies who require chemotherapy.

However, donor T cells may also attack non-tumor host cells, thereby causing Graft Versus Host Disease (GVHD). GVHD is the leading cause of post-HCT complications and can be fatal. Treatment of GVHD may require the use of immunosuppressive therapy or cytotoxicity mediation, which may produce toxicity, result in increased susceptibility to infection or blunt anti-tumor immunity. Early morbidity and mortality associated with acute graft versus host disease (aGVHD) and long-term morbidity associated with chronic gvhd (cgvhd) are major factors limiting HCT success. Both HLA-matched and mismatched transplantation are at risk for GVHD. GVHD can occur even if the HLA of the donor and recipient match, as the immune system can still recognize other differences between its tissues. After allogeneic HCT from HLA-Matched Sibling Donors (MSD), the incidence of aGVHD is between 20% and 60%, even with the use of various immunosuppressive agents.

GVT and GVHD are primarily mediated by conventional T cells (Tcon), which elicit an immune response upon recognition of a cognate antigen by a T cell receptor. Depletion of T cells in HCT grafts may reduce the incidence of GVHD, but may also lead to reduced GVT effects and an increased likelihood of cancer recurrence. Besides Tcon, tregs are another subset of T cells that negatively regulate inflammation and promote immune tolerance. Tregs can prevent or reduce the occurrence of GVHD by their negative regulation of inflammation, including, for example, inflammation caused by donor Tcon recognizing the recipient antigen.

The present invention provides methods for improving HCT comprising administering to a subject certain cell populations including a population comprising HSPCs, a population comprising tregs and a population comprising Tcon. Without being bound by theory, Treg administration may reduce the occurrence of GVHD, while Tcon administration may enhance the GVT effect. Thus, the present invention provides methods of administering the two T cells to enhance the GVT effect while minimizing the occurrence of GVHD. Without being bound by theory, the beneficial aspects of the present invention may be related to the order of cell administration described herein (e.g., administration of tregs prior to Tcon administration) or to the absence of cryopreservation of tregs prior to administration. Thus, the methods disclosed herein can preserve the Graft Versus Tumor (GVT) effect of HCT administration to a cancer subject while preventing or reducing the incidence of Graft Versus Host Disease (GVHD). In some embodiments, two or more cell populations may be administered at different times, e.g., HSPCs and tregs may be administered prior to Tcon administration.

One or more cell populations may be comprised of fresh cells or may be cryopreserved prior to thawing and administration to a subject. Without being bound by theory, administration of fresh tregs may reduce the occurrence of GVHD in a subject, and/or administration of cryopreserved Tcon may reduce the occurrence of GVHD in a subject. For example, administration of cryopreserved Tcon after administration of fresh tregs may reduce the occurrence of GVHD in a subject, increase the GVT effect in a subject, or increase the GVT effect in a subject in the absence of GVHD.

The invention also provides a population of cells comprising tregs that are sorted to produce a population of cells comprising a high percentage of tregs. In some embodiments, administration of a cell population comprising a high percentage of fresh tregs prior to Tcon administration may reduce the occurrence of GVHD in a subject.

Hematopoietic cell transplantation and cancer

The present invention provides methods of subjecting a recipient subject having cancer to hematopoietic stem cell transplantation (HCT). HCT can help treat or alleviate cancer in a subject. In some embodiments, a population of conventional T cells (Tcon) is administered to a subject to elicit a graft-versus-tumor (GVT) immune response.

Patients at high risk of hematological malignancies are rarely cured by standard chemotherapy. For example, high risk malignancies include advanced leukemias and lymphomas after first remission or relapsed refractory leukemias and lymphomas. Myeloablative allogeneic hematopoietic stem cell transplantation (HCT) can increase the survival of the patient, wherein the disease-free survival of the patient is between 10% and 50% and the non-recurrent mortality is between 30% and 50%.

Hematopoietic stem cell transplantation (HCT) refers to the transplantation of multipotent Hematopoietic Stem and Progenitor Cells (HSPCs), typically from bone marrow, peripheral blood or umbilical cord blood. HSPCs have a strong self-renewal capacity and are capable of differentiating into specialized cell types, e.g., are capable of reconstituting all hematopoietic cell lineages. HSPCs can replicate asynchronously, producing two daughter cells with different phenotypes during replication. Hematopoietic stem cells can undergo mitosis and exist in a quiescent form.

HSPCs may be obtained by harvesting from bone marrow or peripheral blood. Bone marrow may be extracted from the posterior or anterior iliac crest while the donor is under local or general anesthesia. HSPCs may be obtained by harvesting from peripheral blood, for example, by peripheral blood apheresis. The number of stem cells harvested can be increased by administering a mobilizing agent (i.e., an agent that mobilizes stem cells from the bone marrow into the peripheral blood) to the donor. Non-limiting examples of mobilizing agents include granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), mozobium, and combinations thereof. Techniques for mobilizing stem cells into peripheral blood may comprise administering a dose of mobilizing agent to a donor, for example, 10 to 40 μ/kg per day. The mobilizing agent may be administered to the donor by several doses of the agent, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. The apheresis product may be separated from the donor about several hours after administration of a dose of the mobilizing agent, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, or 30 hours.

The methods of the invention are useful for treating a human subject having cancer. In some embodiments, the subject has received a cancer treatment, e.g., treatment by a chemotherapeutic drug or radiation therapy. The methods of the invention can help treat hematological malignancies, such as leukemia or lymphoma. Examples of hematologic malignancies that can be treated by the methods described herein include, but are not limited to, Acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), multiple myeloma, and lymphoma (e.g., hodgkin's lymphoma and non-hodgkin's lymphoma). The cancer may be a solid tumor. In some embodiments, the cancer is a primary tumor or a metastatic tumor.

The types of cancer that can be treated using the methods described herein include, but are not limited to, leukemia, lymphoma, adrenocortical cancer, anal cancer, aplastic anemia, cholangiocarcinoma, bladder cancer, bone metastasis, brain cancer, Central Nervous System (CNS) cancer, Peripheral Nervous System (PNS) cancer, breast cancer, cervical cancer, childhood non-hodgkin's lymphoma, colorectal cancer, endometrial cancer, esophageal cancer, ewing's family tumor (e.g., ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal tumor, gestational trophoblastic disease, hairy cell leukemia, hodgkin's lymphoma, kaposi's sarcoma, kidney cancer, larynx and pharynx cancer, acute lymphocytic leukemia, acute myelogenous leukemia, childhood leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, liver cancer, lung carcinoid cancer, biliary cancer, bladder cancer, Male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, cancers of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, melanoma skin cancer, non-melanoma skin cancer, gastric cancer, testicular cancer; thymus cancer, thyroid cancer, uterine cancer (e.g., uterine sarcoma), transitional cell carcinoma, vaginal cancer, vulvar cancer, mesothelioma, squamous cell or epidermoid carcinoma, bronchial adenoma, choriocarcinoma, head and neck cancer, teratocarcinoma, and Waldenstrom's macroglobulinemia.

Hematopoietic cell transplantation and graft-versus-host disease

The present invention provides methods for improving hematopoietic stem cell transplantation (HCT) in which the occurrence of GVHD can be reduced or prevented. In some embodiments, a population of cells described herein is administered to a HCT recipient subject, and the recipient subject does not develop GVHD within a certain period of time (e.g., within 30, 100, or 200 days) after administration of the cells. For example, a population of cells described herein can be administered to a recipient subject of HCT and the recipient subject has a reduced likelihood of developing GVHD within a time period (e.g., within 30, 100, or 200 days) following administration of the cells, relative to a subject receiving HCT according to a different protocol. A population of cells comprising tregs can be administered to a HCT recipient subject, and the population of tregs can reduce or prevent the development of GVHD in the HCT recipient subject. A population of cells comprising fresh tregs may be administered to a HCT recipient subject and may reduce or prevent the development of GVHD in the HCT recipient subject. In some embodiments, a population of cells (e.g., CD4+ FOXP3+ or CD4+ CD25+ CD127dim cells) comprising a high proportion of tregs is administered to a HCT recipient subject and can reduce or prevent the development of GVHD in the HCT recipient subject.

Graft Versus Host Disease (GVHD) is an inflammatory disease that may occur in a xenogeneic transplantation setting. GVHD involves donor cells (transplants) that attack the recipient cells (hosts). GVHD can be divided into acute GVHD (aGVHD) and chronic GVHD (cGVHD). aGVHD usually occurs within the first 3 months after transplantation. aGVHD can be life threatening and can involve the skin, gut and/or liver, etc. cGVHD usually occurs after the first 3 months after transplantation. cGVHD is the leading cause of late treatment-related complications and can be life threatening. In addition to inflammation, cGVHD may also lead to the development of fibrosis, thereby causing functional disorders.

Early morbidity and mortality associated with acute graft versus host disease (aGVHD) and long-term morbidity associated with chronic gvhd (cgvhd) are major factors limiting HCT success. The incidence of aGVHD following allogeneic HCT from HLA-Matched Sibling Donors (MSD) is between 20% and 60%, even when various immunosuppressive agents (e.g., tacrolimus, cyclosporine, methotrexate, mycophenolate, antithymocyte globulin, and corticosteroids) are used. About one third of patients undergoing allogeneic HCT with MSD and non-ex vivo removal of T cell grafts develop chronic GVHD.

GVHD severity was graded, for example, using the Glucksberg grading (I-IV) or the International Bone Marrow Transplant Registry (IBMTR) grading System (A-D). The severity of acute GVHD is determined by assessing skin, liver and gastrointestinal involvement. The stages of individual organ involvement were combined with (Glucksberg) or not (IBMTR) patient physical condition to determine a prognostic overall grade. Grading is important in terms of assessing the response to prevention or treatment, the effect on survival and the relationship to graft versus leukemia effects.

Grade i (a) GVHD is characterized as mild disease; grade ii (b) GVHD is characterized as moderate disease; grade iii (c) GVHD is characterized as severe disease; and grade iv (d) GVHD is characterized as a life-threatening disease. The severity of acute GVHD by the IBMTR classification system is defined as follows: a level: stage 1 simple skin involvement (less than 25% of body surface area present as macular papules), no liver and gastrointestinal tract involvement, stage B: stage 2 skin involvement, stage 1 to stage 2 intestinal or liver involvement, stage C: stage 3 involvement of either organ system (systemic erythroderma; bilirubin 6.1 to 15.0 mg/dL; amount of diarrhea 1500 to 2000 ml/day), stage D: stage 4 involvement of either organ system (systemic erythroderma with bullous formation; bilirubin >15 mg/dL; amount of diarrhea >2000 ml/day, or pain or intestinal obstruction), mortality is significantly higher in patients with moderate to severe GVHD compared to patients with mild disease, e.g., estimated five-year survival of stage iii (C) aGVHD patients is 25%, and estimated five-year survival of stage iv (D) aGVHD patients is 5%.

Treatment of GVHD may require the use of immunosuppressive therapy (e.g., high doses of corticosteroids, chronic administration of immunosuppressive agents) or cytotoxicity mediation, both of which may be toxic. In many cases, immunosuppressive therapy may not be effective in treating GVHD, or result in increased susceptibility to infection, or blunt anti-tumor immunity.

In some embodiments, the disclosed methods prevent or reduce the occurrence of GVHD in a HCT recipient subject. For example, the disclosed methods prevent any pathological manifestations of GVHD in a subject receiving HCT.

For example, the disclosed methods can prevent a subject receiving HCT from developing any GVHD stage 1 or more, any GVHD stage 2 or more, any GVHD stage 3 or more, or any GVHD stage 4.

In some embodiments, the methods disclosed herein can prevent any GVHD in

stage

1, 2, 3, or 4 for at least greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or more in a subject receiving HCT after administration of a population of cells disclosed herein.

The invention provides methods of preventing the appearance of any GVHD at

stage

1, 2, 3 or 4 for no more than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or less in a subject receiving HCT after administration of a population of cells disclosed herein.

For example, the invention provides methods of preventing the development of any GVHD grade a or above, any GVHD grade B or above, any GVHD grade C or above, or any GVHD grade D in a subject receiving HCT.

In some embodiments, the methods described herein can prevent the occurrence of A, B, C or any GVHD grade D for at least greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or more in a subject receiving HCT after administration of a population of cells disclosed herein.

The invention provides methods of preventing the occurrence of A, B, C or any GVHD grade D in a subject receiving HCT for no more than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or less after administration of a population of cells disclosed herein.

For example, the disclosed methods can reduce the proportion of any GVHD occurring in an HCT recipient subject at stage 1 or more, stage 2 or more, stage 3 or more, or stage 4 as compared to other HCT methods.

For example, the methods disclosed herein can reduce the proportion of any GVHD that a HCT recipient subject experiences stage 1 or more, stage 2 or more, stage 3 or more, or stage 4, several days after administration of a population of cells disclosed herein, which days are at least greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or more, as compared to other HCT methods.

In some embodiments, the methods disclosed herein can reduce the proportion of any GVHD occurring at stage 1 or more, stage 2 or more, stage 3 or more, or stage 4 in an HCT recipient subject several days after administration of a population of cells disclosed herein compared to other HCT methods, the days not exceeding about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or less.

For example, the present invention discloses that the proportion of any GVHD occurring at grade a or above, grade B or above, grade C or above or grade D in HCT recipient subjects can be reduced compared to other HCT methods.

For example, the methods disclosed herein can reduce the proportion of any GVHD at or above grade a, grade B or above, grade C or above or grade D in a subject with HCT receptors over a number of days at least greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or above after administration of a population of cells disclosed herein, as compared to other HCT methods.

In some embodiments, the methods disclosed herein can reduce the proportion of any GVHD at or above grade a, grade B or above, grade C or above or grade D in a subject who is a HCT recipient by a number of days not exceeding about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 365, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, 1000 days or below after administration of a population of cells disclosed herein compared to other HCT methods.

Role of immune cells in HCT and GVHD

The present invention provides methods for improving hematopoietic stem cell transplantation (HCT), wherein the methods can reduce or prevent the occurrence of GVHD, as compared to analogous therapeutic methods and the like; wherein the Tcon is not administered after administration of the HSPCs and/or tregs, the tregs are cryopreserved, the Tcon is not cryopreserved, or any combination thereof. Subpopulations of immune cells (e.g., conventional T cells (Tcon), regulatory T cells (Treg), constant natural killer T cells (iNKT), and memory T cells (Tmem)) may have an effect on GVHD that occurs after HCT, but may also have an effect on such things as GVT immune response, immune reconstitution, susceptibility to infection, and survival.

Methods comprising administering a population of Tcon and Treg to a HCT recipient subject are disclosed. In some embodiments, iNKT is also administered. In some embodiments, Tmem is also administered. The invention provides parameters for administration of the cell population that contribute to a good clinical prognosis of the HCT receptor subject. Without being bound by theory, parameters that may be helpful to a HCT recipient subject to obtain a good clinical prognosis include, for example, the population administered, the order and timing of administration of the different populations, the purity criteria of the populations, the method of obtaining the populations, the method of processing or storage of the populations (e.g., whether fresh or frozen cell populations are used), the dose of the populations administered, the method of obtaining the populations, and combinations thereof.

GVHD is primarily mediated by donor T cells, which elicit an inflammatory response upon recognition of the recipient antigen. T Cell Depletion (TCD) of the donor graft can be achieved to reduce the likelihood of acute and chronic GVHD. T cell depletion can be achieved by methods including, but not limited to, physical adsorption of T cells onto protein ligands (e.g., lectins), immunodepletion using T cell-specific antibodies, and immunoaffinity techniques (e.g., using T cell or lymphocyte-specific antibodies, Magnetic Activated Cell Sorting (MACS), or Fluorescence Activated Cell Sorting (FACS) in immunoadsorption chromatography columns). Application of TCD technology to donor grafts increased T cell depletion by 10-fold to 10-fold⁵Doubling and reducing the incidence of GVHD. However, TCD may also lead to an increased incidence of cancer recurrence, as the lack of T cells may attenuate the graft-versus-tumor (GVT) immune response. In addition, TCD may also lead to impaired immune recovery and increased susceptibility to infection.

GVT and GVHD are primarily mediated by conventional T cells (Tcon), which elicit an immune response upon recognition of a cognate antigen by a T cell receptor (a tumor antigen for GVT; a non-tumor receptor antigen for GVHD). For example, Tcon may have an effect on GVT, GVHD or a combination thereof. Without being bound by theory, administration of Tcon after Treg administration may enhance GVT immunity, and/or reduce susceptibility to infection.

Tcon, as used herein, refers broadly to all CD3+ T cells, cells expressing medium to high levels of CD127, cells expressing CD3 and medium to high levels of CD127, or cells expressing CD3, medium to high levels of CD127, and CD4 or CD 8. In some embodiments, Tcon does not express V α 24J α 18 TCR. Tcon and Treg may be non-mutually exclusive cell populations. In some embodiments, Tcon and Treg are mutually exclusive cell populations.

Regulatory T cells ("tregs") are a special subset of T cells that suppress activation of the immune system, thereby maintaining immune tolerance. Without being bound by theory, the population of tregs administered by the methods of the invention may contribute to a good clinical prognosis, for example by reducing or preventing the development of GVHD in a transplant recipient and/or improving immune reconstitution in a transplant recipient. For example, administration of Treg and HSPCs prior to conventional T cell (Tcon) administration helps to preserve the graft-versus-tumor (GVT) effect and prevent GVHD. For example, administration of Treg and HSPCs prior to conventional T cell (Tcon) administration helps to preserve the graft-versus-tumor (GVT) effect and reduce the incidence of GVHD relative to other HCT approaches. For example, administration of Treg and HSPCs prior to conventional T cell (Tcon) administration helps to preserve the graft-versus-tumor (GVT) effect and reduce the risk of developing GVHD relative to other HCT approaches. Without being bound by theory, for example, administration of Treg may prevent GVHD, while administration of Tcon may increase GVT effects relative to other HCT methods. In some embodiments, Treg administration may reduce the risk of developing GVHD, while Tcon administration may increase GVT effects relative to other HCT methods.

There are various types of tregs, e.g., TCR α β + CD4+ regulatory T cells, including natural regulatory T cells (ntregs) and induced regulatory T cells (itrregs). ntregs are produced in the thymus and transmitted to peripheral T cells as a long-lived lineage of autoantigen-specific lymphocytes. iTreg recruits self-circulating lymphocytes, which acquire regulatory properties under specific peripheral stimulatory conditions. nTreg and iTreg are CD4+ CD25 +; both inhibited proliferation of CD4+ CD25-T cells in a dose-dependent manner, and both were anergic and did not proliferate under TCR stimulation. In addition to being positive for CD4 and CD25, tregs are also positive for the transcription factor FOXP3, an intracellular marker. Tregs can be identified or selected based on various marker expression profiles. Non-limiting examples of marker expression profiles that can be used to select tregs include (1) CD4+ CD25+ CD127 dim; (2) CD4+ FOXP3 +; (3) CD3+ CD4+ CD25 +; (5) CD3+ CD4+ CD25+ CD127 dim; (6) CD3+ CD4+ CD25+ CD127dim FOXP3 +; (7) CD3+ FOXP3 +; (8) CD3+ CD4+ FOXP3 +; (9) CD3+ CD4+ CD25+ FOXP3 +; (10) CD3+ CD25+ FOXP3 +; (11) CD3+ CD25+ CD127 dim; (12) CD4+ CD25 +; (13) CD4+ CD25+ CD127dimFOXP3 +; (14) FOXP3+, CD4+ FOXP3 +; (15) CD4+ CD25+ FOXP3 +; (16) CD25+ FOXP3 +; or (17) CD25+ CD127 dim.

Selection based on certain expression profiles can be achieved based on extracellular markers without permeabilizing the cells, e.g., selection based on CD4+ CD25+ CD127 dim.

For example, in the methods described herein, administration of tregs to a subject receiving HCT can help prevent graft rejection, help prevent GVHD, help reduce the occurrence of GVHD, promote hematopoietic reconstitution, promote immune reconstitution, promote mixed chimerism, or a combination thereof.

In the methods of the invention, a subject may be administered constant natural killer T cells (inkts). iNKT is a subset of CD1 d-restricted natural killer T cells (NKTs) that express a highly conserved α β -T cell receptor consisting of a V α 24J α 18TCR α chain (referred to herein as "V α 24J α 18") in humans⁺"). iNKT cells can be identified by binding to CD1 d-multimers, e.g., CD1 d-multimers loaded with alpha-galactosylceramide (GalCer), PBS-57, PBS-44, or other natural or synthetic glycolipids, which can also be tetramers, dendrimers, other structures, Fc fusions, or their fusion counterpartsAny combination thereof. Another method of identification is an antibody or combination of antibodies that specifically recognizes the V α 24J α 18 region. Examples include the V α 24 antibody, the J α 18 antibody, or the monoclonal antibody clone 6B11 that specifically binds to a unique region of the V α 24J α 18TCR and can be used to identify iNKT cells. The iNKT may be CD3⁺Vα24Jα18⁺。

For example, in the methods described herein, administration of iNKT to a subject receiving HCT can promote transplantation, increase GVT effects, reduce the incidence of GVHD, reduce susceptibility to cancer recurrence, reduce susceptibility to infection, or a combination thereof. In some embodiments, iNKT may increase the activity of tregs. In some embodiments, iNKT may increase the activity of HSPCs.

In the methods described herein, a memory T cell (Tmem) may be administered to the subject. Tmem may refer to antigen-contacted T cells that express, among other things, phenotypic markers such as CD45RO, TCR α, TCR β, CD3, CD4, or CD8, CD95, and IL-2R β. The memory T cells have immunity and can be in an inactive state for a long time. Memory T cells can rapidly acquire effector functions after being re-primed with antigen. The memory T cell population may include any combination of subsets of central memory T cells and effector memory T cells. In some embodiments, Tmem is CD3+ CD45RA-CD45RO +. For example, in the methods described herein, administration of Tmem to a subject receiving HCT can increase the GVT effect, reduce the incidence of GVHD, reduce the susceptibility to recurrence of cancer, reduce the susceptibility to infection, or a combination thereof.

HLA type and donor/host matching

The present invention provides methods comprising administering a population of cells to a subject. The population of cells to be administered to the recipient may be from an allogeneic donor. Donor and recipient can be HLA typed to determine whether any of their HLA alleles match.

Human Leukocyte Antigens (HLA), also widely referred to as Major Histocompatibility Complex (MHC) antigens, are protein molecules expressed on the surface of cells that can confer antigenicity to the cells. HLA/MHC antigens are target molecules that can be recognized by T cells and Natural Killer (NK) cells as hematopoietic stem cells from the same source as immune effector cells ("self"), or as hematopoietic cells from another source ("non-self"). HLA class I antigens (in humans, A, B and C are included) are expressed by most cells, while HLA class II antigens (in humans, DR, DP and DQ are included) are mainly expressed on professional antigen presenting cells. Both HLA classes are associated with GVHD.

HLA antigens are encoded by highly polymorphic genes; a series of alleles exist for each HLA class I and class II gene. The allelic products differ in one or more amino acids in the alpha and/or beta domains. Specific antibody or nucleic acid agent sets are used to haplotype the HLA of an individual using leukocytes expressing class I and class II molecules. HLA alleles can be described at various levels of detail. Most names begin with HLA-and locus names, followed by a number x and some number (even number) of numbers representing alleles. The first two digits represent a set of alleles. Previous typing methods often fail to completely distinguish between alleles and therefore remain at this level. The third four digits represent synonymous alleles. The fifth six digit represents any synonymous mutation within the coding framework of the gene. The seventh eight digit number distinguishes mutations outside the coding region. Letters such as L, N, Q or S may be followed by allele names to indicate expression levels or other known relevant non-genomic data. Thus, the number of digits for a fully described allele may be up to 9, excluding HLA prefixes and locus notations.

Three HLA genes are considered to be the most important genes for HLA typing of potential transplant donors and recipients, namely HLA-A, HLA-B and HLA-DR. Since human subjects carry two copies of each of the genes, there are six genes in total that are HLA-typed genes.

HLA-matched refers to a donor-recipient pair in which the six HLA-A, HLA-B and HLA-DR alleles are all matched between the donor and recipient. HLA mismatch refers to a donor-recipient pair having at least one HLA antigen mismatch between the donor and recipient. GVHD can occur even if the HLA of the donor and recipient match, as the immune system can still recognize other differences between its tissues.

The HLA allelic genome inherited from a certain parent forms a haplotype. Determining the patient's haplotype can help predict the probability of finding a matching donor and help in developing a search strategy, as some alleles and haplotypes are more common than others and they are distributed with different frequencies in different ethnic and ethnic groups. HLA haploidy match is a donor-recipient pair having a chromosome match between the donor and recipient of at least HLA-A, HLA-B and HLA-DR. Haploid concordance pairs may or may not match on other alleles, e.g., other HLA genes on the other chromosome, or other histocompatibility loci on either chromosome. The donor is typically from a family member, e.g., a father (or mother) that haploids with a child; and possibly haploid concordant siblings.

The present invention provides methods comprising administering a population of cells to a subject. The population of cells to be administered to the recipient may be from an allogeneic donor.

In some embodiments, the disclosed cell populations are administered to a recipient subject after HLA-typing the donor and recipient, e.g., determining whether the HLA-match or HLA-mismatch of the donor and recipient is determined by typing HLA alleles HLA-A, HLA-B and HLA-DR of the donor and recipient. The HLA's of the donor-recipient pairs of the present invention may be matched, mismatched or haploid. There may be a relationship between the donor and the recipient (e.g., parent, daughter, sibling, grandparent, grandchild, aunt, uncle, or epigenetic). There may be a relationship between the donor and recipient, which may be HLA matched, HLA mismatched or haploid matched. There may be no relationship between the donor and the acceptor.

Fitting together

Following administration of the donor-derived transplanted cells to a recipient, the chimerism in the recipient can be monitored. Chimerism may refer to the mixing of donor cells with host cells in an individual receiving HCT. Patients who are mixed, but not fully chimerized, are at significantly reduced risk of developing GVHD and, therefore, achieving mixed chimerism is desirable. Furthermore, higher frequency of immunodeficiency and infection was observed with complete chimerism compared to mixed chimerism. In some embodiments, the methods provided herein allow a human subject to achieve mixed chimerism.

Complete donor-type chimerism refers to a subject showing over 95% of donor cells in a given cell lineage at any time after transplantation. Mixed chimerism refers to more than 1% but less than 95% of the donor DNA in a subject in the assay. Subjects exhibiting mixed chimerism can be further classified according to chimerism evolution, wherein improving mixed chimerism can comprise continuously increasing the proportion of donor cells over a period of at least 6 months. Stable mixed chimerism can involve fluctuating percentages of recipient cells over time without complete loss of donor cells.

Whether a subject is a fully chimeric, mixed chimeric, or non-chimeric is determined by analyzing a sample of hematopoietic cells (e.g., peripheral blood or bone marrow) from a graft recipient. The analysis can be performed by any practical typing method. In some embodiments, PCR with microsatellite probes is used to periodically monitor the degree of chimerism between all monocytes, T cells, B cells, CD56+ NK cells and CD15+ neutrophils. For example, commercially available kits can be used to quantify donor and host genetic material extracted from cells based on short terminal repeat length polymorphisms. The automated reader provides the percentage of donor type cells according to the corresponding standard curve for the artificial donor and host cell mixture.

Method of producing a composite material

Collecting cells

One or more cell populations of the invention may be obtained from a single donor, for example, by an mobilized peripheral blood apheresis of a single donor. HSPCs, tregs, Tcon, inkts, Tmem, or any combination thereof may be obtained from a single donor.

One or more cell populations of the invention may be obtained from one donor and one or more other cell populations of the invention may be obtained from another donor. One cell population of the invention may be obtained from a single donor, and another cell population of the invention may be obtained from multiple donors.

The populations of the invention may be obtained from multiple donors, for example, by mobilized peripheral blood apheresis from multiple donors. HSPCs may be obtained from multiple donors. Tregs can be obtained from multiple donors. Tcon may be obtained from multiple donors. inkts can be obtained from multiple donors. Tmem can be obtained from multiple donors.

The donor and recipient of the present invention may be allogeneic. The donor and recipient HLA of the present invention may be matched. The donor and recipient HLA of the present invention may be mismatched, for example, at 1, 2, 3, 4, 5 or 6 major HLA alleles. The donor and recipient of the present invention may be haploid concordant.

In some embodiments, the cell population of the present invention can be obtained from whole blood. The cell populations of the invention can be obtained from peripheral blood apheresis products, e.g., mobilized peripheral blood apheresis products. The cell populations of the invention may be obtained from at least one apheresis product, two apheresis products, three apheresis products, four apheresis products, five apheresis products, six apheresis products, or more apheresis products.

In some embodiments, the cell population of the invention may be obtained from bone marrow. In some embodiments, the cell population of the invention can be obtained from umbilical cord blood.

Cell processing

The cell population of the invention can be refined by selecting from another cell population (e.g., peripheral blood or a peripheral blood apheresis product). The method of selecting a cell population may comprise a method involving positive or negative selection of a cell population of interest. The method of selecting a cell population may comprise an affinity reagent including, but not limited to, an antibody, a full length antibody, an antibody fragment, a natural antibody, a synthetic antibody, an engineered antibody, a full length affibody, an affibody fragment, a full length avidin, an avidin fragment, a full length anticalin, an anticalin fragment, a full length avimer, an avimer fragment, a full length DARPin, a DARPin fragment, a full length fynomer, a fynomer fragment, a full length kunitz domain peptide, a kunitz domain peptide fragment, a full length monomer, a monomer fragment, a peptide, or a polyamino acid. In some embodiments, the affinity reagent is directly coupled to the detection reagent and/or the purification reagent. In some cases, the detection reagent and the purification reagent are the same. In other cases, the detection reagent and the purification reagent are different. For example, the detection reagent and/or purification reagent is a fluorescent reagent, a magnetic reagent, or the like. In some cases, the detection reagent and/or purification reagent is magnetic particles for column purification. For example, magnetic chromatography column purification of chromatography columns, antibodies, buffers, preparative materials and reagents can be performed using the Miltenyi system (CliniMACs)

The affinity reagent may comprise an immunoaffinity reagent that utilizes the binding specificity of the antibody or fragment or derivative thereof to positively or negatively select a population of cells of interest. The method of selection of the cell population may comprise affinity agents and chromatography columns, for example, Magnetic Activated Cell Sorting (MACS) using specific antibodies and microbeads. The method of selecting the cell population may comprise Fluorescence Activated Cell Sorting (FACS) based on a staining profile obtained using one or more fluorescence conjugated antibodies to sort the cell population. The method of selecting a cell population may comprise physisorption, for example, by T cells onto a protein ligand (e.g., a lectin).

The HSPC populations of the present invention may be selected based on the expression of CD 34. For example, the HSPC populations of the present invention may be selected using anti-CD 34 antibodies as part of a Magnetic Activated Cell Sorting (MACS) or Fluorescence Activated Cell Sorting (FACS) system.

For example, the number of HPSCs in a cell population can be determined by flow cytometry to quantify CD34+ cells. In some embodiments, the dose calculation results can be adjusted based on a measure of cell viability determination, e.g., by flow cytometry (using propidium iodide or 7-AAD) or by trypan blue stain exclusion,

the population of cells comprising tregs of the invention may be selected based on the expression of markers including CD3, CD4, CD25, CD127, FOXP3, and combinations thereof.

Populations of cells comprising tregs may be selected using Magnetic Activated Cell Sorting (MACS). Populations of cells comprising tregs may be selected using Fluorescence Activated Cell Sorting (FACS). The population of cells comprising tregs may be selected by multiple procedures, e.g., multiple MACS selections, multiple FACS selections, or a combination of MACS and FACS selections. For example, a first selection can be made to express CD25, e.g., using MACS to isolate CD25+ cells from a sample of hematopoietic cells. The second selection may be performed by contacting CD25+ cells with antibodies specific for CD4 and CD127, wherein CD4+ CD127dim cells may be isolated using FACS.

A population of cells comprising tregs may be isolated from whole blood. Cell populations comprising tregs can be isolated from peripheral blood apheresis products. A population of cells comprising tregs may be isolated from a population of cells that were previously enriched and/or depleted for one or more other cell types, for example, from a population of cells depleted of CD34+ cells. In some embodiments, tregs are isolated from CD34+ MACS sorted flow-through fractions.

For example, the number of tregs in a cell population can be determined by flow cytometry, where tregs can be identified as such as CD4+ CD25+ CD127dim or CD4+ FOXP3 +. The dose calculation results can be adjusted based on a measure of cell viability determination, e.g., by flow cytometry (using propidium iodide or 7-AAD) or by trypan blue stain exclusion,

the Tcon population may be derived from peripheral blood. The Tcon population may be derived from peripheral blood apheresis products.

In some embodiments, no selection step is performed and the Tcon population is derived directly from peripheral blood or an aliquot of apheresis product. In some embodiments, the cell population may be enriched for Tcon, e.g., by sorting based on expression of various markers, using MACS, FACS, or a combination thereof. The Tcon population can be enriched by sorting CD3+ cells. The Tcon population can be enriched by sorting CD4+ and CD8+ cells. The Tcon population may be enriched by negative selection, wherein non-Tcon cells are removed, e.g., by MACS depletion of cells expressing CD34, CD19, CD25, or a combination thereof.

For example, the number of Tcon present in the population can be quantified by flow cytometry to quantify CD3+ cells. The number of CD3+ cells in an aliquot can be determined, as well as the volume of CD3 cells comprising the appropriate dose administered to the recipient. The dose calculation results can be adjusted based on a measure of cell viability, for example, by flow cytometry (using propidium iodide or 7-AAD) or by trypan blue staining exclusion.

The apheresis product of the invention may be divided into two parts, one part for providing Tcon cells and the other part for separating and purifying HSPCs and tregs. In some embodiments, CD34+ cells are isolated from the apheresis product and purified to produce a CD 34-negative cell fraction from which tregs can be isolated.

The iNKT population may be derived from peripheral blood. The iNKT population may be derived from peripheral blood apheresis products.

The cell population can be enriched for inkts, e.g., by sorting based on expression of various markers, using MACS, FACS, or a combination thereof. For example, iNKT populations may be sorted by CD3⁺Vα24Jα18⁺And (4) enriching cells.

For example, the number of inkts present in a population may be quantified by flow cytometry for CD3⁺Vα24Jα18⁺Cells were quantified. Determination of CD3 in aliquots⁺Vα24Jα18⁺The number of cells, and determining the volume containing the appropriate dose of iNKT to be administered to the recipient. In some embodiments, the dose calculation results may be adjusted based on a measure of cell viability determination, for example, viability determined by flow cytometry (using propidium iodide or 7-AAD) or by trypan blue stain exclusion.

The Tmem population may be derived from peripheral blood. The Tmem population may be derived from peripheral blood apheresis products.

The cell population can be enriched for Tmem, for example, by sorting based on expression of various markers, using MACS, FACS, or a combination thereof. For example, the Tmem population may be enriched by sorting CD3+ CD45RA-CD45RO + cells.

For example, the amount of Tmem present in a population can be quantified by flow cytometry to quantify CD3+ CD45RA-CD45RO + cells. The number of CD3+ CD45RA-CD45RO + cells in an aliquot can be determined, as well as the volume containing the appropriate dose of Tmem to be administered to the recipient. The dose calculation results can be adjusted based on a measure of cell viability determination, for example, by flow cytometry (using propidium iodide or 7-AAD) or by trypan blue staining exclusion.

Using freshly or cryopreserved cells

The cell populations described herein may be administered in a fresh state after isolation, or after cryopreservation and subsequent thawing.

Cells freshly isolated from a donor ("fresh cells") can be administered to a recipient subject. Fresh cells can be stored in a buffer, for example, CliniMACS PBS-EDTA buffer containing 0.5% human serum albumin, or pH 7.4Plasma-Lyte-A containing 2% human serum albumin. Fresh cells can be stored at low temperatures (e.g., 2-8 ℃) but must not be cryopreserved/frozen.

After obtaining a population of fresh cells from a donor, the fresh cells can be stored for a period of at least greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 120, 96, 150, 300, or more hours prior to administration to a subject.

After obtaining a population of fresh cells from a donor, the fresh cells can be stored for a period of time of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 120, 150, 300, or less hours prior to administration to a subject.

The cells of the invention may be cryopreserved. In some embodiments, cryopreservation may be beneficial for the methods disclosed herein. For example, cryopreservation of Tcon may reduce the occurrence of GVHD prior to subsequent thawing and administration to a subject.

Cryopreservation may involve the addition of a preservative (e.g., DMSO) and gradual cooling of the cells in a controlled rate freezer to prevent damage to cell permeability during the formation of the ice crystals. Cryopreservation may include commercially available cryopreservation reagents and materials, e.g., cold packs and

CS10。

cryopreserved cells can be stored at low temperatures for hours to years. Cryopreserved cells can be stored at ultra-low temperatures, for example, -50 ℃, -60 ℃, -70 ℃, -80 ℃, -90 ℃, -100 ℃, -110 ℃, -120 ℃, -130 ℃, -140 ℃, -150 ℃, -160 ℃, -170 ℃, -180 ℃, -190 ℃, -196 ℃ or less. Cryopreserved cells can be stored in a storage device containing liquid nitrogen.

The cells can be cryopreserved before or after the other methods described herein are performed, e.g., before or after the sorting method is performed, before or after the characterization method is performed (e.g., determining cell viability or cell concentration of a particular type).

In some embodiments, whole blood may be cryopreserved. Whole blood can be cryopreserved without sorting or characterization. Whole blood can be cryopreserved after sorting but without characterization; whole blood can be cryopreserved after characterization but without sorting. Whole blood can be cryopreserved after characterization and sorting. Whole blood may be cryopreserved after quantification of one cell type according to the present invention. Whole blood may be cryopreserved after quantification of conventional T cells (Tcon, e.g., CD3+ cells). Whole blood may be cryopreserved after quantifying viability of all or a population of cells (e.g., conventional T cells) described herein.

The peripheral blood apheresis products of the invention can be cryopreserved. Peripheral blood apheresis products can be cryopreserved without sorting or characterization. Peripheral blood apheresis products can be cryopreserved after sorting but without characterization. Peripheral blood apheresis products can be cryopreserved after characterization but without sorting. Peripheral blood apheresis products can be cryopreserved after characterization and sorting. The peripheral blood apheresis product may be cryopreserved after quantification of one of the cell types described herein. Peripheral blood apheresis products may be cryopreserved after quantification of conventional T cells (Tcon, e.g., CD3+ cells). Peripheral blood apheresis products can be cryopreserved after quantifying viability of all or a population of cells described herein (e.g., conventional T cells).

A cell population selected or selected from another cell population can be cryopreserved, for example, a population of HSPCs, tregs, tcons, inkts, or tmems can be cryopreserved.

The cells of the invention may be cryopreserved for any period of time. Cells described herein can be cryopreserved for a number of hours prior to thawing and administration to a subject, the time being at least greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours or more.

Cells described herein can be cryopreserved for several hours prior to thawing and administration to a subject, the time not exceeding about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours or less.

Cells of the invention can be cryopreserved for a number of days at least greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 or more days before thawing and administering to a subject.

Cells of the invention can be cryopreserved for several days no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 or less days prior to thawing and administration to a subject.

Cell administration

Sequence and timing of administration

The present invention provides methods for enhancing allogeneic hematopoietic stem cell transplantation comprising administering a population of cells to a subject.

In some embodiments, a population of Hematopoietic Stem Progenitor Cells (HSPCs), a population of cells comprising regulatory T cells (tregs), and a population of conventional T cells (Tcon) are administered to a subject.

The HSPC population and the Treg-containing cell population can be administered at the same or similar time or at different times.

The HSPC population and the cell population comprising tregs may be administered up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours apart.

The HSPC population may be administered to the subject prior to the Tcon population.

The population of Tcon may be administered several hours after the subject is administered the HSPC population, for at least greater than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

In some embodiments, the subject may be administered the HSPC population several hours before the Tcon population, which time is no more than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

The subject may be administered the HSPC population several hours prior to the administration of the Tcon population, e.g., in the time range of about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 24-42, 24-36, 24-72, 24-42, 24-36, or a combination thereof, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours.

The subject may be administered a population of cells comprising tregs prior to the administration of a population of Tcon.

The Tcon population may be administered several hours after the subject is administered a population of cells comprising tregs for at least greater than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

In some embodiments, the Tcon population may be administered several hours after the subject is administered a population of cells comprising tregs, said time not exceeding about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

The Tcon population may be administered several hours after the Treg population is first administered to the subject, e.g., within the time range of about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 24-42, 24-36-72, 24-36, 24-60, 24-42, 24-36, 24-72, or a combination thereof, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours.

The HSPC population and the cell population comprising tregs may be administered to the subject prior to the Tcon population.

The Tcon population may be administered several hours after the subject is administered the HSPC population and the cell population comprising tregs, e.g., for at least greater than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

In some embodiments, the population of HSPCs and the population of cells comprising tregs may be administered to the subject several hours prior to the administration of the population of Tcon, which time is no more than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

The population of HSPCs and the population of cells comprising tregs may be administered to the subject several hours after the previous administration of the population of HSPCs and the population of cells comprising tregs, e.g., in the range of about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours.

In some embodiments, a population of Hematopoietic Stem Progenitor Cells (HSPCs), a population of cells comprising regulatory T cells (tregs), a population of conventional T cells (Tcon), and a population of constant natural killer T cells (inkts) are administered to the subject.

The iNKT population and the HSPC population may be administered to the subject at the same time or at similar times. In some embodiments, the subject is administered the HSPC population prior to the iNKT population.

The iNKT population may be administered several hours after the HSPC population is administered to the subject, at least greater than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

In some embodiments, the iNKT population is administered several hours after the HSPC population is administered to the subject and the time is not more than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

The iNKT population may be administered several hours after the HSPC population is administered to the subject, e.g., in the time range of about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 24-42, 24-36, 24-72, 24-60, 24-42, 24-36, or 24-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours.

The iNKT population and the population of cells comprising tregs may be administered to the subject at the same time or at similar times. In some embodiments, the population of cells comprising tregs is administered to the subject prior to the administration of the iNKT population.

The iNKT population may be administered several hours after the subject is administered a population of cells comprising tregs, said time being at least greater than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

In some embodiments, the iNKT population is administered several hours after the subject is previously administered a population of cells comprising tregs, the time not exceeding about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

The iNKT population may be administered several hours after the Treg population is first administered to the subject, e.g., within a time range of about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 24-42, 24-36-72, 24-36, 24-42, 24-36-72, or a combination thereof, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours.

In some embodiments, a population of Hematopoietic Stem Progenitor Cells (HSPCs), a population of cells comprising regulatory T cells (tregs), a population of conventional T cells (Tcon), and a population of memory T cells (Tmem) are administered to the subject.

The Tmem population and the HSPC population can be administered to the subject at the same time or at similar times. In some embodiments, the subject is administered the HSPC population prior to the Tmem population.

The Tmem population may be administered several hours after the HSPC population is administered to the subject, at least greater than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

In some embodiments, the Tmem population is administered several hours after the HSPC population is administered to the subject, no more than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

The Tmem population may be administered several hours after the HSPC population is administered to the subject, e.g., in the range of about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 24-42, 24-36, 24-72, 24-42, 24-36, or 24-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours.

The population of Tmem and the population of cells comprising tregs may be administered to the subject at the same time or at similar times. In some embodiments, the population of cells comprising tregs is administered to the subject prior to the population of Tmem.

The population of Tmem may be administered several hours after the subject is first administered a population of cells comprising tregs for at least greater than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

In some embodiments, the Tmem population is administered several hours after the subject is previously administered a population of cells comprising tregs, the time not exceeding about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours.

The population of Tmem may be administered several hours after the subject is first administered a population of tregs, e.g., within the time range of about 6-96, 12-84, 12-72, 12-66, 12-60, 12-54, 12-48, 12-42, 12-36, 12-30, 12-24, 12-18, 18-72, 18-66, 18-60, 18-54, 18-48, 18-42, 18-36, 18-30, 18-24, 24-72, 24-66, 24-60, 24-54, 24-48, 24-42, 24-36, 24-30, 30-72, 30-66, 30-60, 30-54, 30-48, 30-42, 30-36, 36-72, 24-42, 24-36, 24-48, 24-42, 24-36, or 24-72, 36-66, 36-60, 36-54, 36-48, 36-42, 42-72, 42-66, 42-60, 42-54, 42-48, 48-72, 48-66, 48-60, 48-54, 54-72, 54-66, 54-60, 60-72, 60-66, or 66-72 hours.

Dosage form

HSPC

The population of HPSCs can be administered to the subject in a dose of at least greater than about 1 x 10 per kilogram body weight of the subject⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or more than one cell.

In some embodiments, the population of HPSCs can be administered to the subject in a dose that does not exceed about 1 x 10 per kilogram of body weight of the subject⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or the following cells.

The HPSC population may be administered to the subject in a dose, e.g., at least greater than about 1X 10 per kilogram body weight of the subject⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or the above CD34+ cells.

The HPSC population may be administered to the subject in a dose not exceeding about 1X 10 per kilogram body weight of the subject⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or the following CD34+ cells.

For example, the HPSC population may be administered to a subject at a dose ranging from 1 x 10 per kilogram of body weight of the subject⁴To 1X 10⁹、1×10⁵To 1X 10⁸、1×10⁵To 2X 10⁷、5×10⁵To 2X 10⁷、5×10⁵To 1.5X 10⁷、5×10⁵To 1X 10⁷、5×10⁵To 9X 10⁶、5×10⁵To 8X 10⁶、5×10⁵To 7X 10⁶、5×10⁵To 6X 10⁶、5×10⁵To 5X 10⁶、5×10⁵To 4X 10⁶、5×10⁵To 3X 10⁶、5×10⁵To 2X 10⁶、5×10⁵To 1X 10⁶、1×10⁶To 1.5X 10⁷、1×10⁶To 1X 10⁷、1×10⁶To 9X 10⁶、1×10⁶To 8X 10⁶、1×10⁶To 7X 10⁶、1×10⁶To 6X 10⁶、1×10⁶To 5X 10⁶、1×10⁶To 4X 10⁶、1×10⁶To 3X 10⁶、1×10⁶To 2X 10⁶、1.5×10⁶To 1.5X 10⁷、1.5×10⁶To 1X 10⁷、1.5×10⁶To 9X 10⁶、1.5×10⁶To 8X 10⁶、1.5×10⁶To 7X 10⁶、1.5×10⁶To 6X 10⁶、1.5×10⁶To 5X 10⁶、1.5×10⁶To 4X 10⁶、1.5×10⁶To 3X 10⁶、1.5×10⁶To 2X 10⁶、2×10⁶To 1.5X 10⁷、2×10⁶To 1X 10⁷、2×10⁶To 9X 10⁶、2×10⁶To 8X 10⁶、2×10⁶To 7X 10⁶、2×10⁶To 6X 10⁶、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2.5×10⁶To 1.5X 10⁷、2.5×10⁶To 1X 10⁷、2.5×10⁶To 9X 10⁶、2.5×10⁶To 8X 10⁶、2.5×10⁶To 7X 10⁶、2.5×10⁶To 6X 10⁶、2.5×10⁶To 5X 10⁶、2.5×10⁶To 4X 10⁶Or 2.5X 10⁶To 3X 10⁶And (4) cells.

The HPSC population may be administered to the subject in a dose, for example, in the range of 1X 10 per kilogram body weight of the subject⁴To 1X 10⁹、1×10⁵To 1X 10⁸、1×10⁵To 2X 10⁷、5×10⁵To 2X 10⁷、5×10⁵To 1.5X 10⁷、5×10⁵To 1X 10⁷、5×10⁵To 9X 10⁶、5×10⁵To 8X 10⁶、5×10⁵To 7X 10⁶、5×10⁵To 6X 10⁶、5×10⁵To 5X 10⁶、5×10⁵To 4X 10⁶、5×10⁵To 3X 10⁶、5×10⁵To 2X 10⁶、5×10⁵To 1X 10⁶、1×10⁶To 1.5X 10⁷、1×10⁶To 1X 10⁷、1×10⁶To 9X 10⁶、1×10⁶To 8X 10⁶、1×10⁶To 7X 10⁶、1×10⁶To 6X 10⁶、1×10⁶To 5X 10⁶、1×10⁶To 4X 10⁶、1×10⁶To 3X 10⁶、1×10⁶To 2X 10⁶、1.5×10⁶To 1.5X 10⁷、1.5×10⁶To 1X 10⁷、1.5×10⁶To 9X 10⁶、1.5×10⁶To 8X 10⁶、1.5×10⁶To 7X 10⁶、1.5×10⁶To 6X 10⁶、1.5×10⁶To 5 is10⁶、1.5×10⁶To 4X 10⁶、1.5×10⁶To 3X 10⁶、1.5×10⁶To 2X 10⁶、2×10⁶To 1.5X 10⁷、2×10⁶To 1X 10⁷、2×10⁶To 9X 10⁶、2×10⁶To 8X 10⁶、2×10⁶To 7X 10⁶、2×10⁶To 6X 10⁶、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2.5×10⁶To 1.5X 10⁷、2.5×10⁶To 1X 10⁷、2.5×10⁶To 9X 10⁶、2.5×10⁶To 8X 10⁶、2.5×10⁶To 7X 10⁶、2.5×10⁶To 6X 10⁶、2.5×10⁶To 5X 10⁶、2.5×10⁶To 4X 10⁶Or 2.5X 10⁶To 3X 10⁶And CD34+ cells.

The HSPC populations of the present invention may have defined levels of CD34+ cell purity.

For example, a HSPC population described herein may comprise a proportion of CD34+ cells in the total cell, nucleated cell, or CD45+ cell count that is at least greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

In some embodiments, the HSPC population of the present invention comprises a proportion of CD34+ cells in the total cell, nucleated cell, or CD45+ cell count that is no more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less.

The HSPC populations of the present invention may have defined levels of contaminating CD3+ cells.

For example, CD3+ cells are present in the HSPC population of the present invention in an amount such that at least greater than about 1 x 10 CD3+ cells are obtained per kilogram body weight of the receptor²、2×10²、3×10²、4×10²、5×10²、6×10²、7×10²、8×10²、9×10²、1×10³、2×10³、3×10³、4×10³、5×10³、6×10³、7×10³、8×10³、9×10³、1×10⁴、2×10⁴、3×10⁴、4×10⁴、5×10⁴、6×10⁴、7×10⁴、8×10⁴、9×10⁴、1×10⁵Or more.

In some embodiments, CD3+ cells are present in the HSPC population of the present invention in an amount such that no more than about 1 x 10 CD3+ cells are obtained per kilogram body weight of the receptor²、2×10²、3×10²、4×10²、5×10²、6×10²、7×10²、8×10²、9×10²、1×10³、2×10³、3×10³、4×10³、5×10³、6×10³、7×10³、8×10³、9×10³、1×10⁴、2×10⁴、3×10⁴、4×10⁴、5×10⁴、6×10⁴、7×10⁴、8×10⁴、9×10⁴、1×10⁵Or the following.

The HSPC population of the present invention may comprise a proportion of CD3+ cells in the total cell, nucleated cell, or CD45+ cell count of at least greater than about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 3.9%, 3.3%, 3.8%, 3.6%, 3.5%, 3.8%, 3.6%, 3.8%, 3.7%, 3.8%, 3.6%, 3.8%, 3%, 3.6%, 3.8%, 3%, 3.8%, 3.

In some embodiments, the HSPC population of the present invention comprises a proportion of CD3+ cells in the total cell, nucleated cell, or CD45+ cell count that is no more than about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.9%, 3.3%, 3.5%, 3.8%, 3.9%, 3.5%, 3.6%, 3.3.3.5%, 3.3.6%, 3.3.5%, 3%, 3.8%, 3.5%, 3.8%, 3%, 3.7%, 3.8%, 3%, 3.8%, 3%, 3.7%, 3%, 3.8%, or less.

Treg

The population of cells comprising tregs may be administered to the subject at a dose of at least greater than about 1 x 10 per kilogram body weight of the subject⁴、1×10⁵、2×10⁵、3×10⁵、4×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or more than one cell.

In some embodiments, a population of cells comprising tregs may be administered to a subject at a dose of no more than about 1 x 10 per kilogram body weight of the subject⁴、1×10⁵、2×10⁵、3×10⁵、4×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or the following cells.

The population of cells comprising tregs may be administered to the subject at a dose, for example, at least greater than about 1 x 10 per kilogram of body weight of the subject⁵、2×10⁵、3×10⁵、4×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or a Treg of the above, wherein the Treg is defined as CD4+ CD25+ CD127dim, CD3+ CD4+ CD25+, CD3+ CD4+ CD25+ CD127dim, CD3+ CD4+ CD25+ CD127dim FOXP3+, CD3+ FOXP3+, CD3+ CD4+ FOXP3+, CD3+ CD4+ CD25+ FOXP 25+, CD25+ FOXP 25+, CD25+ CD25+ CD 36127 dim, CD25+ CD25+ FOXP 25+, CD25+ FOXP 25+ CD25+ CD 36127 dim.

The population of cells comprising tregs may be administered to the subject at a dose, e.g., no more than about 1 x 10 per kilogram of body weight of the subject⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or a Treg defined as CD4+ CD25+ CD127dim, CD3+ CD4+ CD25+, CD3+ CD4+ CD25+ CD127dim, CD3+ CD4+ CD25+ CD127dim FOXP3+, CD3+ FOXP3+, CD3+ CD4+ FOXP3+, CD3+ CD4+ CD25+ FOXP 25+, CD25+ FOXP 25+, CD25+ CD 36127 dim, CD25+, CD25+ FOXP 25+ CD 36127 dim.

The population of cells comprising tregs may be administered to the subject in a dose ranging from 1 x 10 per kilogram body weight of the recipient⁴To 1X 10⁹、1×10⁵To 1X 10⁸、1×10⁵To 1X 10⁷、5×10⁵To 1X 10⁷、5×10⁵To 5X 10⁶、5×10⁵To 4X 10⁶、5×10⁵To 3X 10⁶、5×10⁵To 2X 10⁶、5×10⁵To 1X 10⁶、1×10⁶To 1X 10⁷、1×10⁶To 5X 10⁶、1×10⁶To 4X 10⁶、1×10⁶To 3X 10⁶、1×10⁶To 2X 10⁶、1×10⁶To 1.5X 10⁶、1.5×10⁶To 1X 10⁷、1.5×10⁶To 5X 10⁶、1.5×10⁶To 4X 10⁶、1.5×10⁶To 3X 10⁶、1.5×10⁶To 2X 10⁶、2×10⁶To 1X 10⁷、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2×10⁶To 1X 10⁷、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2×10⁶To 2X 10⁶、2.5×10⁶To 1X 10⁷、2.5×10⁶To 5X 10⁶、2.5×10⁶To 4X 10⁶Or 2.5X 10⁶To 3X 10⁶And (4) cells.

In some embodiments, a population of cells comprising tregs may be administered to a subject at a dose ranging from 1 x 10 per kilogram body weight of the recipient⁴To 1X 10⁹、1×10⁵To 1X 10⁸、1×10⁵To 1X 10⁷、5×10⁵To 1X 10⁷、5×10⁵To 5X 10⁶、5×10⁵To 4X 10⁶、5×10⁵To 3X 10⁶、5×10⁵To 2X 10⁶、5×10⁵To 1X 10⁶、1×10⁶To 1X 10⁷、1×10⁶To 5X 10⁶、1×10⁶To 4X 10⁶、1×10⁶To 3X 10⁶、1×10⁶To 2X 10⁶、1×10⁶To 1.5X 10⁶、1.5×10⁶To 1X 10⁷、1.5×10⁶To 5X 10⁶、1.5×10⁶To 4X 10⁶、1.5×10⁶To 3X 10⁶、1.5×10⁶To 2X 10⁶、2×10⁶To 1X 10⁷、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2×10⁶To 1X 10⁷、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2×10⁶To 2X 10⁶、2.5×10⁶To 1X 10⁷、2.5×10⁶To 5X 10⁶、2.5×10⁶To 4X 10⁶Or 2.5X 10⁶To 3X 10⁶A Treg, wherein the Treg is defined as CD4+ CD25+ CD127dim, CD3+ CD4+ CD25+, CD3+ CD4+ CD25+ CD127dim, CD3+ CD4+ CD25+ CD127dim FOXP3+, CD3+ FOXP3+, CD3+ CD4+ FOXP3+, CD3+ CD4+ CD25+ FOXP 25+, CD25+ CD 36127 dim, CD25+, CD25+ FOXP 25+ CD 36127 dim, CD25+ FOXP 25+ CD 36127 dim.

A population of tregs-containing cells of the invention can comprise FOXP3+ cells in a proportion of the total, nucleated, or CD45+ cells, e.g., at least greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

In some embodiments, a population of tregs-containing cells described herein comprises FOXP3+ cells in a proportion of no more than about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less of the total cell, nucleated cell, or CD45+ cell count.

A population of tregs-containing cells of the invention can comprise a proportion of CD4+ CD25+ CD127dim cells in the total cell, nucleated cell, or CD45+ cell count, e.g., at least greater than about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less.

In some embodiments, a population of tregs-containing cells described herein comprises CD4+ CD25+ CD127dim cells in a proportion of the total cell, nucleated cell, or CD45+ cell count that is no more than about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less.

For example, a population of cells comprising tregs according to the invention may comprise a proportion of tregs in the total cell, nucleated cell or CD45+ cell count that is at least greater than about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, wherein tregs are defined as CD4+ CD25+ CD127dim, CD3+ CD4+ CD25+, CD3+ CD4+ 25+ CD127dim, CD 36 + CD4+ CD25+ CD 36127 dim, CD 73738 + FOXP + 4+ CD 38 + FOXP + 3+ 5+ FOXP + 5+ CD 36 + (iii + FOXP3+ CD 6326 + (iii + CD) CD3+ CD4+ CD25+ FOXP3+, CD3+ CD25+ FOXP3+, CD3+ CD25+ CD127dim, CD4+ CD25+, CD4+ CD25+ CD127dim FOXP3+, FOXP3+, CD4+ FOXP3+, CD4+ CD25+ FOXP3+, CD25+ FOXP3+ or CD25+ CD127 dim.

In some embodiments, a population of cells comprising tregs described herein comprises a proportion of tregs in the total cell, nucleated cell, or CD45+ cell count that is no more than about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less, wherein tregs are defined as CD4+ CD25+ CD127dim, CD3+ CD4+ CD25+, CD3+ CD4+ 25+ CD127dim, CD 36 + 4+ CD25+ CD 36127 dim, CD 38 + FOXP 38 + 4+ CD 3946 +, CD 36 + FOXP3+ 3946 +, CD 6326 + FOXP + CD 6326 + CD 38 + FOXP 2 + CD 38 + CD 3975 +, CD 38 + FOXP, CD3+ CD4+ CD25+ FOXP3+, CD3+ CD25+ FOXP3+, CD3+ CD25+ CD127dim, CD4+ CD25+, CD4+ CD25+ CD127dim FOXP3+, FOXP3+, CD4+ FOXP3+, CD4+ CD25+ FOXP3+, CD25+ FOXP3+ or CD25+ CD127 dim.

The population of tregs-comprising cells of the invention can comprise FOXP3+ cells in a proportion in the number of nucleated cells or CD45+ cells, e.g., in the range of 50% to 100%, 60% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 81% to 100%, 82% to 100%, 83% to 100%, 84% to 100%, 86% to 100%, 87% to 100%, 88% to 100%, 89% to 100%, 90% to 91%, 92% to 100%, 93% to 100%, 94% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 99.5% to 100%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, 81% to 99%, 82% to 99%, 83% to 99%, 84% to 99%, 85% to 99%, 86% to 99%, 87% to 99%, or a combination thereof, 88% to 99%, 89% to 99%, 90% to 99%, 91% to 99%, 92% to 99%, 94% to 99%, 95% to 99%, 96% to 97%, 98% to 99%, 50% to 98%, 60% to 98%, 70% to 98%, 80% to 98%, 81% to 98%, 82% to 98%, 83% to 98%, 84% to 98%, 85% to 98%, 86% to 98%, 87% to 98%, 88% to 98%, 89% to 98%, 90% to 98%, 91% to 98%, 92% to 98%, 94% to 98%, 95% to 98%, 96% to 97%, 98% to 98%, 50% to 97%, 60% to 97%, 70% to 97%, 80% to 97%, 81% to 97%, 82% to 97%, 83% to 97%, 84% to 97%, 85% to 97%, 86% to 97%, 87% to 97%, 88% to 97%, 89% to 97%, 90% to 97%, 70% to 97%, 80% to 97%, 92% to 98%, 80%, 94% to 98%, 98% to 97%, or more, 91% to 97%, 92% to 97%, 94% to 97%, 95% to 97%, 96% to 97%, 50% to 96%, 60% to 96%, 70% to 96%, 80% to 96%, 81% to 96%, 82% to 96%, 83% to 96%, 84% to 96%, 85% to 96%, 86% to 96%, 87% to 96%, 88% to 96%, 89% to 96%, 90% to 96%, 91% to 96%, 92% to 96%, 94% to 96%, 95% to 96%, 50% to 95%, 60% to 95%, 70% to 95%, 80% to 95%, 81% to 95%, 82% to 95%, 83% to 95%, 84% to 95%, 85% to 95%, 86% to 95%, 87% to 95%, 88% to 95%, 89% to 95%, 90% to 95%, 91% to 95%, 92% to 95%, or 94% to 95%.

A population of cells comprising tregs of the invention can comprise tregs in a proportion in the total cell, nucleated cell, or CD45+ cell number that ranges from 50% to 100%, 60% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 81% to 100%, 82% to 100%, 83% to 100%, 84% to 100%, 86% to 100%, 87% to 100%, 88% to 100%, 89% to 100%, 90% to 91%, 92% to 100%, 93% to 100%, 94% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 99.5% to 100%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, 81% to 99%, 82% to 99%, 83% to 99%, 84% to 99%, 85% to 99%, 86% to 99%, 87% to 99%, 88% to 99%, 89% to 99%, 90% to 99%, 91% to 99%, 92% to 99%, 94% to 99%, 95% to 99%, 96% to 97%, 98% to 99%, 50% to 98%, 60% to 98%, 70% to 98%, 80% to 98%, 81% to 98%, 82% to 98%, 83% to 98%, 84% to 98%, 85% to 98%, 86% to 98%, 87% to 98%, 88% to 98%, 89% to 98%, 90% to 98%, 91% to 98%, 92% to 98%, 94% to 98%, 95% to 98%, 96% to 97%, 98% to 98%, 50% to 97%, 60% to 97%, 70% to 97%, 80% to 97%, 81% to 97%, 82% to 97%, 83% to 97%, 84% to 97%, 85% to 97%, 86% to 97%, 87% to 97%, 88% to 97%, 89% to 97%, 90% to 97%, 70% to 97%, 80% to 97%, 92% to 98%, 80%, 94% to 98%, 98% to 97%, or more, 91% to 97%, 92% to 97%, 94% to 97%, 95% to 97%, 96% to 97%, 50% to 96%, 60% to 96%, 70% to 96%, 80% to 96%, 81% to 96%, 82% to 96%, 83% to 96%, 84% to 96%, 85% to 96%, 86% to 96%, 87% to 96%, 88% to 96%, 89% to 96%, 90% to 96%, 91% to 96%, 92% to 96%, 94% to 96%, 95% to 96%, 50% to 95%, 60% to 95%, 70% to 95%, 80% to 95%, 81% to 95%, 82% to 95%, 83% to 95%, 84% to 95%, 85% to 95%, 86% to 95%, 87% to 95%, 88% to 95%, 89% to 95%, 90% to 95%, 91% to 95%, 92% to 95%, or 94% to 95%, wherein Treg is defined as CD4+ CD25+ CD19 + 539 2 + 3+, CD3+ CD4+ CD25+ CD127dim, CD3+ CD4+ CD25+ CD127dim FOXP3+, CD3+ FOXP3+, CD3+ CD4+ FOXP3+, CD3+ CD4+ CD25+ FOXP3+, CD3+ CD25+ FOXP3+, CD3+ CD25+ CD127dim, CD4+ CD25+, CD4+ CD25+ CD127dim FOXP3+, FOXP3+, CD4+ FOXP3+, CD4+ CD25+ FOXP3+, CD25+ FOXP3+ or CD25+ CD127 dim.

The population of cells comprising tregs of the invention may have a defined level of contaminating non-Treg cells.

For example, non-Treg cells are present in the population of Treg-containing cells of the invention in an amount such that at least greater than about 1 x 10 non-Treg cells are obtained per kilogram body weight of the recipient²、2×10²、3×10²、4×10²、5×10²、6×10²、7×10²、8×10²、9×10²、1×10³、2×10³、3×10³、4×10³、5×10³、6×10³、7×10³、8×10³、9×10³、1×10⁴、2×10⁴、3×10⁴、4×10⁴、5×10⁴、6×10⁴、7×10⁴、8×10⁴、9×10⁴、1×10⁵Or above, wherein the non-Treg cells are FOXP 3-or CD127 +/bright.

In some embodiments, non-Treg cells are present in the population of Treg-containing cells of the invention in an amount such that no more than about 1 x 10 non-Treg cells are obtained per kilogram body weight of the recipient²、2×10²、3×10²、4×10²、5×10²、6×10²、7×10²、8×10²、9×10²、1×10³、2×10³、3×10³、4×10³、5×10³、6×10³、7×10³、8×10³、9×10³、1×10⁴、2×10⁴、3×10⁴、4×10⁴、5×10⁴、6×10⁴、7×10⁴、8×10⁴、9×10⁴、1×10⁵Or wherein the non-Treg cells are FOXP 3-or CD127 +/bright.

The population of cells comprising tregs of the invention may comprise a proportion of non-Treg cells in total cells, nucleated cells or CD45+ cells, for example, the ratio is at least greater than about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.6%, 3.8%, 3.9%, 3.8%, 3.1%, 3.2%, 3.3.3.3.3%, 3.3.3.4%, 3%, 3.6%, 3.7%, 3.8%, 3%, 8%, 10%, or more, wherein the non-Treg cells are FOXP 3-or CD127 +/bright.

In some embodiments, the population of cells comprising tregs of the invention comprises a proportion of non-Treg cells in the total cell, nucleated cells or CD45+ cell count, the ratio is not more than about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 8%, 3.9%, 3.7%, 8%, 3.9%, 3.1%, 3.3.3.3.3%, 3.3.2%, 3.3.3.3%, 3.5%, 3.6%, 3.7%, 8%, 3%, 8%, 7%, 8, wherein the non-Treg cells are FOXP 3-or CD127 +/bright.

Tcon

The population of tcons can be administered to a subject in a dose, e.g., at least greater than about 1 x 10 per kilogram body weight of the subject⁴、1×10⁵、2×10⁵、3×10⁵、4×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or more than one cell.

In some embodiments, a population of tcons may be administered to a subject at a dose of no more than about 1 x 10 per kilogram of body weight of the subject⁴、1×10⁵、2×10⁵、3×10⁵、4×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or the following cells.

The population of tcons may be administered to a subject in a dose of at least greater than about 1 x 10 per kilogram body weight of the subject⁵、2×10⁵、3×10⁵、4×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or more Tcon, where Tcon is defined as CD3 +.

The population of tcons may be administered to a subject in a dose not exceeding about 1 x 10 per kilogram body weight of the subject⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or Tcon, where Tcon is defined as CD3 +.

In some embodiments, a population of tcons may be administered to a subject at a dose ranging from 1 x 10 per kilogram of body weight of the subject⁴To 1X 10⁹、1×10⁵To 1X 10⁸、1×10⁵To 1X 10⁷、5×10⁵To 1X 10⁷、5×10⁵To 5X 10⁶、5×10⁵To 4X 10⁶、5×10⁵To 3X 10⁶、5×10⁵To 2X 10⁶、5×10⁵To 1X 10⁶、1×10⁶To 1X 10⁷、1×10⁶To 5X 10⁶、1×10⁶To 4X 10⁶、1×10⁶To 3X 10⁶、1×10⁶To 2X 10⁶、1×10⁶To 1.5X 10⁶、1.5×10⁶To 1X 10⁷、1.5×10⁶To 5X 10⁶、1.5×10⁶To 4X 10⁶、1.5×10⁶To 3X 10⁶、1.5×10⁶To 2X 10⁶、2×10⁶To 1X 10⁷、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2×10⁶To 1X 10⁷、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2×10⁶To 2X 10⁶、2.5×10⁶To 1X 10⁷、2.5×10⁶To 5X 10⁶、2.5×10⁶To 4X 10⁶Or 2.5X 10⁶To 3X 10⁶And (4) cells.

In some embodiments, a population comprising a Tcon may be administered to a subject at a dose ranging from 1 x 10 per kilogram body weight of the subject⁴To 1X 10⁹、1×10⁵To 1X 10⁸、1×10⁵To 1X 10⁷、5×10⁵To 1X 10⁷、5×10⁵To 5X 10⁶、5×10⁵To 4X 10⁶、5×10⁵To 3X 10⁶、5×10⁵To 2X 10⁶、5×10⁵To 1X 10⁶、1×10⁶To 1X 10⁷、1×10⁶To 5X 10⁶、1×10⁶To 4X 10⁶、1×10⁶To 3X 10⁶、1×10⁶To 2X 10⁶、1×10⁶To 1.5X 10⁶、1.5×10⁶To 1X 10⁷、1.5×10⁶To 5X 10⁶、1.5×10⁶To 4X 10⁶、1.5×10⁶To 3X 10⁶、1.5×10⁶To 2X 10⁶、2×10⁶To 1X 10⁷、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2×10⁶To 1X 10⁷、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2×10⁶To 2X 10⁶、2.5×10⁶To 1X 10⁷、2.5×10⁶To 5X 10⁶、2.5×10⁶To 4X 10⁶Or 2.5X 10⁶To 3X 10⁶A Tcon, wherein Tcon is defined as CD3 +.

A population of tcons described herein may comprise a proportion of CD3+ cells in the total, nucleated, or CD45+ cell count that is at least greater than about 5%, 10%, 20%, 30%, 40%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

A population of tcons described herein may comprise a proportion of CD3+ cells in the total, nucleated, or CD45+ cell count of no more than about 5%, 10%, 20%, 30%, 40%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less.

For example, a population of tcons described herein may comprise CD3+ cells in a proportion in the total cell, nucleated cell, or CD45+ cell count range from 10% to 100%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 100%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 100%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 50%, 30% to 40%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 100%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, or a percentage of CD45+, 60% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 100%, 70% to 90%, 70% to 80%, 80% to 100%, 80% to 90%, or 90% to 100%.

iNKT

The iNKT population can be administered to a subject at a dose of at least greater than about 1 x 10 per kilogram body weight of the subject⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or more than one cell.

The iNKT population can be administered to a subject at a dose that does not exceed about 1X 10 per kilogram body weight of the subject⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or the following cells.

In some embodiments, the iNKT population can be administered to a subject at a dose of at least greater than about 1 x 10 per kilogram of body weight of the receptor⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or more than one CD3⁺Vα24Jα18⁺A cell.

In some embodiments, the iNKT population can be administered to a subject at a dose that does not exceed about 1 x 10 per kilogram of body weight of the receptor⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or the following CD3⁺Vα24Jα18⁺A cell.

The iNKT population can be administered to the subject in a dose, e.g., in a range of 1X 10 per kilogram of body weight of the subject⁴To 1X 10⁹、1×10⁵To 1X 10⁸、1×10⁵To 2X 10⁷、5×10⁵To 2X 10⁷、5×10⁵To 1.5X 10⁷、5×10⁵To 1X 10⁷、5×10⁵To 9X 10⁶、5×10⁵To 8X 10⁶、5×10⁵To 7X 10⁶、5×10⁵To 6X 10⁶、5×10⁵To 5X 10⁶、5×10⁵To 4X 10⁶、5×10⁵To 3X 10⁶、5×10⁵To 2X 10⁶、5×10⁵To 1X 10⁶、1×10⁶To 1.5X 10⁷、1×10⁶To 1X 10⁷、1×10⁶To 9X 10⁶、1×10⁶To 8X 10⁶、1×10⁶To 7X 10⁶、1×10⁶To 6X 10⁶、1×10⁶To 5X 10⁶、1×10⁶To 4X 10⁶、1×10⁶To 3X 10⁶、1×10⁶To 2X 10⁶、1.5×10⁶To 1.5X 10⁷、1.5×10⁶To 1X 10⁷、1.5×10⁶To 9X 10⁶、1.5×10⁶To 8X 10⁶、1.5×10⁶To 7X 10⁶、1.5×10⁶To 6X 10⁶、1.5×10⁶To 5X 10⁶、1.5×10⁶To 4X 10⁶、1.5×10⁶To 3X 10⁶、1.5×10⁶To 2X 10⁶、2×10⁶To 1.5X 10⁷、2×10⁶To 1X 10⁷、2×10⁶To 9X 10⁶、2×10⁶To 8X 10⁶、2×10⁶To 7X 10⁶、2×10⁶To 6X 10⁶、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2.5×10⁶To 1.5X 10⁷、2.5×10⁶To 1X 10⁷、2.5×10⁶To 9X 10⁶、2.5×10⁶To 8X 10⁶、2.5×10⁶To 7X 10⁶、2.5×10⁶To 6X 10⁶、2.5×10⁶To 5X 10⁶、2.5×10⁶To 4X 10⁶Or 2.5X 10⁶To 3X 10⁶And (4) cells.

The iNKT population can be administered to a subject in a dose range of 1X 10 per kilogram of body weight of the recipient⁴To 1X 10⁹、1×10⁵To 1X 10⁸、1×10⁵To 2X 10⁷、5×10⁵To 2X 10⁷、5×10⁵To 1.5X 10⁷、5×10⁵To 1X 10⁷、5×10⁵To 9X 10⁶、5×10⁵To 8X 10⁶、5×10⁵To 7X 10⁶、5×10⁵To 6X 10⁶、5×10⁵To 5X 10⁶、5×10⁵To 4X 10⁶、5×10⁵To 3X 10⁶、5×10⁵To 2X 10⁶、5×10⁵To 1X 10⁶、1×10⁶To 1.5X 10⁷、1×10⁶To 1X 10⁷、1×10⁶To 9X 10⁶、1×10⁶To 8X 10⁶、1×10⁶To 7X 10⁶、1×10⁶To 6X 10⁶、1×10⁶To 5X 10⁶、1×10⁶To 4X 10⁶、1×10⁶To 3X 10⁶、1×10⁶To 2X 10⁶、1.5×10⁶To 1.5X 10⁷、1.5×10⁶To 1X 10⁷、1.5×10⁶To 9X 10⁶、1.5×10⁶To 8X 10⁶、1.5×10⁶To 7X 10⁶、1.5×10⁶To 6X 10⁶、1.5×10⁶To 5X 10⁶、1.5×10⁶To 4X 10⁶、1.5×10⁶To 3X 10⁶、1.5×10⁶To 2X 10⁶、2×10⁶To 1.5X 10⁷、2×10⁶To 1X 10⁷、2×10⁶To 9X 10⁶、2×10⁶To 8X 10⁶、2×10⁶To 7X 10⁶、2×10⁶To 6X 10⁶、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2.5×10⁶To 1.5X 10⁷、2.5×10⁶To 1X 10⁷、2.5×10⁶To 9X 10⁶、2.5×10⁶To 8X 10⁶、2.5×10⁶To 7X 10⁶、2.5×10⁶To 6X 10⁶、2.5×10⁶To 5X 10⁶、2.5×10⁶To 4X 10⁶Or 2.5X 10⁶To 3X 10⁶An individual CD3⁺Vα24Jα18⁺A cell.

Tmem

The population of Tmem can be administered to a subject at a dose of at least greater than about 1 x 10 per kilogram of body weight of the subject⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or more than one cell.

The population of Tmem may be administered to a subject at a dose not exceeding about 1 x 10 per kilogram body weight of the subject⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or the following cells.

For example, a dose can be administered to a subjectAdministering a Tmem population at a dose of at least greater than about 1 x 10 per kilogram body weight of the subject⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or more than one CD3⁺CD45RA^-CD45RO⁺A cell.

In some embodiments, the population of Tmem may be administered to the subject at a dose not exceeding about 1 x 10 per kilogram body weight of the subject⁴、1×10⁵、5×10⁵、6×10⁵、7×10⁵、8×10⁵、9×10⁵、1×10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、1×10⁷、1.5×10⁷、2×10⁷、2.5×10⁷、3×10⁷、3.5×10⁷、4×10⁷、4.5×10⁷、5×10⁷、5.5×10⁷、6×10⁷、6.5×10⁷、7×10⁷、7.5×10⁷、8×10⁷、8.5×10⁷、9×10⁷、9.5×10⁷、1×10⁸、1×10⁸、1.5×10⁸、2×10⁸、2.5×10⁸、3×10⁸、3.5×10⁸、4×10⁷、4.5×10⁸、5×10⁸、5.5×10⁸、6×10⁸、6.5×10⁸、7×10⁸、7.5×10⁸、8×10⁸、8.5×10⁸、9×10⁸、9.5×10⁸、1×10⁹Or the following CD3⁺CD45RA^-CD45RO⁺A cell.

In some embodiments, the population of Tmem may be administered to the subject at a dose ranging from 1 x 10 per kilogram of body weight of the subject⁴To 1X 10⁹、1×10⁵To 1X 10⁸、1×10⁵To 2X 10⁷、5×10⁵To 2X 10⁷、5×10⁵To 1.5X 10⁷、5×10⁵To 1X 10⁷、5×10⁵To 9X 10⁶、5×10⁵To 8X 10⁶、5×10⁵To 7X 10⁶、5×10⁵To 6X 10⁶、5×10⁵To 5X 10⁶、5×10⁵To 4X 10⁶、5×10⁵To 3X 10⁶、5×10⁵To 2X 10⁶、5×10⁵To 1X 10⁶、1×10⁶To 1.5X 10⁷、1×10⁶To 1X 10⁷、1×10⁶To 9X 10⁶、1×10⁶To 8X 10⁶、1×10⁶To 7X 10⁶、1×10⁶To 6X 10⁶、1×10⁶To 5X 10⁶、1×10⁶To 4X 10⁶、1×10⁶To 3X 10⁶、1×10⁶To 2X 10⁶、1.5×10⁶To 1.5X 10⁷、1.5×10⁶To 1X 10⁷、1.5×10⁶To 9X 10⁶、1.5×10⁶To 8X 10⁶、1.5×10⁶To 7X 10⁶、1.5×10⁶To 6X 10⁶、1.5×10⁶To 5X 10⁶、1.5×10⁶To 4X 10⁶、1.5×10⁶To 3X 10⁶、1.5×10⁶To 2X 10⁶、2×10⁶To 1.5X 10⁷、2×10⁶To 1X 10⁷、2×10⁶To 9X 10⁶、2×10⁶To 8X 10⁶、2×10⁶To 7X 10⁶、2×10⁶To 6X 10⁶、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2.5×10⁶To 1.5X 10⁷、2.5×10⁶To 1X 10⁷、2.5×10⁶To 9X 10⁶、2.5×10⁶To 8X 10⁶、2.5×10⁶To 7X 10⁶、2.5×10⁶To 6X 10⁶、2.5×10⁶To 5X 10⁶、2.5×10⁶To 4X 10⁶Or 2.5X 10⁶To 3X 10⁶And (4) cells.

The population of Tmem may be administered to the subject in a dose, for example, in the range of 1 x 10 per kilogram body weight of the subject⁴To 1X 10⁹、1×10⁵To 1X 10⁸、1×10⁵To 2X 10⁷、5×10⁵To 2X 10⁷、5×10⁵To 1.5X 10⁷、5×10⁵To 1X 10⁷、5×10⁵To 9X 10⁶、5×10⁵To 8X 10⁶、5×10⁵To 7X 10⁶、5×10⁵To 6X 10⁶、5×10⁵To 5X 10⁶、5×10⁵To 4X 10⁶、5×10⁵To 3X 10⁶、5×10⁵To 2X 10⁶、5×10⁵To 1X 10⁶、1×10⁶To 1.5X 10⁷、1×10⁶To 1X 10⁷、1×10⁶To 9X 10⁶、1×10⁶To 8X 10⁶、1×10⁶To 7X 10⁶、1×10⁶To 6X 10⁶、1×10⁶To 5X 10⁶、1×10⁶To 4X 10⁶、1×10⁶To 3X 10⁶、1×10⁶To 2X 10⁶、1.5×10⁶To 1.5X 10⁷、1.5×10⁶To 1X 10⁷、1.5×10⁶To 9X 10⁶、1.5×10⁶To 8X 10⁶、1.5×10⁶To 7X 10⁶、1.5×10⁶To 6X 10⁶、1.5×10⁶To 5X 10⁶、1.5×10⁶To 4X 10⁶、1.5×10⁶To 3X 10⁶、1.5×10⁶To 2X 10⁶、2×10⁶To 1.5X 10⁷、2×10⁶To 1X 10⁷、2×10⁶To 9X 10⁶、2×10⁶To 8X 10⁶、2×10⁶To 7X 10⁶、2×10⁶To 6X 10⁶、2×10⁶To 5X 10⁶、2×10⁶To 4X 10⁶、2×10⁶To 3X 10⁶、2.5×10⁶To 1.5X 10⁷、2.5×10⁶To 1X 10⁷、2.5×10⁶To 9X 10⁶、2.5×10⁶To 8X 10⁶、2.5×10⁶To 7X 10⁶、2.5×10⁶To 6X 10⁶、2.5×10⁶To 5X 10⁶、2.5×10⁶To 4X 10⁶Or 2.5X 10⁶To 3X 10⁶An individual CD3⁺CD45RA^-CD45RO⁺A cell.

Treg to Tcon ratio

For example, in the methods described herein, the ratio of Tcon to Treg administered to a subject may be about 1:100, 1:50, 1:25, 1:20, 1:15, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2.5, 1:2, 1.5:2, 1:1.5, 1:1, 1.5:1, 2:1.5, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 50:1, or 100: 1.

Pretreatment regimens, GVHD prophylactic agents, and other agents.

In some embodiments of the methods described herein, the subject may be treated with a myeloablative conditioning prior to infusion of the cell populations described herein.

In some embodiments, the methods of the invention may comprise administering one or more immunosuppressive or other agents to the subject.

For example, immunosuppressive agents may be administered to a subject to prevent rejection of a transplant by the recipient immune system, or to prevent or reduce GVHD caused by the transplant attacking the recipient cells. Immunosuppressants include, for example, calcineurin inhibitors (which bind to binding proteins to inhibit calcineurin activity), monoclonal antibodies, and the like.

Calcineurin inhibitors include, for example, tacrolimus, cyclosporine a, and the like. Adjuvants may be administered in combination with calcineurin inhibitors and include, for example, steroids, azathioprine, mycophenolate mofetil, sirolimus, and the like. Monoclonal antibodies useful as immunosuppressive agents include, for example, antagonist antibodies (e.g., antibodies that antagonize IL-2R, such as basiliximab and daclizumab) and antibodies that deplete immune cell populations through antibody-dependent cellular cytotoxicity (e.g., anti-CD 3 antibodies that deplete T cells, such as muromonab-CD3), and the like

The methods of the invention may further comprise administering to a subject in need of a relevant treatment an anti-neoplastic agent, or a pharmaceutically acceptable salt or prodrug thereof. Examples of antineoplastic agents include, but are not limited to, antineoplastic alkylating agents, antineoplastic antimetabolites, antineoplastic antibiotics, plant-derived antineoplastic agents, antineoplastic organo-platinum compounds, antineoplastic camptothecin derivatives, antineoplastic tyrosine kinase inhibitors, monoclonal antibodies, interferons, biological response modifiers, and other agents having antineoplastic activity or pharmaceutically acceptable salts thereof.

Exemplary embodiments

In some embodiments, the present invention discloses a method of treating a human subject in need of treatment described herein, comprising administering to the human subject: a) a population of Hematopoietic Stem Progenitor Cells (HSPC), wherein the HSPC population comprises more than 1 x 10 per kilogram body weight of the human subject⁵HSPC (preferably, 5X 10)⁵To 2X 10⁷HSPC), wherein the HPSC is CD34⁺(ii) a b) A cell population comprising regulatory T cells (Tregs), wherein the Tregs are CD4⁺CD25⁺CD127^dimOr CD4⁺FOXP3⁺Wherein the population of cells comprising Tregs comprises more than 1 x 10 per kilogram body weight of the human subject⁵Treg (preferably, 5 × 10)⁵To 4X 10⁶A population of tregs), wherein said population of tregs-containing cells has not been cryopreserved prior to said administration thereof; and c) a conventional T cell (Tcon) population, wherein the Tcon is CD3+, wherein the Tcon population comprises less than 1 x 10 weight per kilogram body weight of the human subject⁷Tcon (preferably, 5 × 10)⁵To 4X 10⁶A Tcon), wherein said HSPC population and said cell population comprising tregs are administered prior to (preferably 36-60 hours prior to) administration of said Tcon population.

In some embodiments, the present invention discloses a method of treating a human subject in need of treatment described herein, comprising administering to the human subject: a) a population of Hematopoietic Stem Progenitor Cells (HSPC), wherein the HSPC population comprises more than 1 x 10 per kilogram body weight of the human subject⁵HSPC (preferably, 5X 10)⁵To 2X 10⁷HSPC), wherein the HPSC is CD34⁺(ii) a b) A cell population comprising regulatory T cells (Tregs), wherein the Tregs are CD4⁺CD25⁺CD127^dimOr CD4⁺FOXP3⁺Wherein the population of cells comprising Tregs comprises more than 1 x 10 per kilogram body weight of the human subject⁵Treg (preferably, 5 × 10)⁵To 4X 10⁶A population of tregs), wherein said population of tregs-containing cells has not been cryopreserved prior to said administration thereof; c) a conventional T cell (Tcon) population, wherein the Tcon is CD3+, wherein the Tcon population comprises less than 1 x 10 per kilogram body weight of the human subject⁷Tcon (preferably, 5 × 10)⁵To 4X 10⁶Tcon); and d) a Graft Versus Host Disease (GVHD) prophylactic agent (preferably tacrolimus or sirolimus), wherein said HSPC population and said population of Treg-containing cells are administered prior to (preferably, 36-60 hours prior to) the administration of said Tcon population, and wherein said human subject does not develop Graft Versus Host Disease (GVHD) within 30 days (preferably, within 1 year) of said administration of said Tcon populationDisease (GVHD).

In some embodiments, the present invention discloses a method of treating a human subject in need of treatment described herein, comprising administering to the human subject: a) a population of Hematopoietic Stem Progenitor Cells (HSPCs), wherein the HSPC population comprises more than 1 x 10 per kilogram body weight of the human subject⁵HSPC (preferably, 5X 10)⁵To 2X 10⁷HSPC), wherein the HPSC is CD34⁺(ii) a b) A cell population comprising regulatory T cells (tregs), wherein said tregs are CD4⁺CD25⁺CD127^dimOr CD4⁺FOXP3⁺Wherein the population of cells comprising Tregs comprises more than 1 x 10 per kilogram body weight of the human subject⁵Treg (preferably, 5 × 10)⁵To 4X 10⁶A Treg), wherein the population of cells comprising tregs comprises CD45⁺Cells in which more than 50% (preferably, more than 90%) of said CD45⁺The cells are tregs, wherein said population of cells comprising tregs has not been cryopreserved prior to said administration thereof; and c) a conventional T cell (Tcon) population, wherein said Tcon is CD3+, wherein said Tcon population comprises less than 1 x 10 per kilogram body weight of said human subject⁷Tcon (preferably, 5 × 10)⁵To 4X 10⁶A Tcon), wherein said Tcon population is cryopreserved prior to said administration thereof, wherein said HSPC population and said Treg-comprising cell population are administered prior to (preferably 36-60 hours prior to) administration of said Tcon population.

In some embodiments, the present invention discloses a method of treating a human subject in need of treatment described herein, comprising administering to the human subject: a) a population of Hematopoietic Stem Progenitor Cells (HSPC), wherein the HSPC population comprises more than 1 x 10 per kilogram body weight of the human subject⁵HSPC (preferably, 5X 10)⁵To 2X 10⁷HSPC), wherein the HPSC is CD34⁺(ii) a b) A cell population comprising regulatory T cells (Tregs), wherein the Tregs are CD4⁺CD25⁺CD127^dimOr CD4⁺FOXP3⁺Wherein the population of cells comprising Tregs comprisesMore than 1 x 10 per kilogram body weight of said human subject⁵Treg (preferably, 5 × 10)⁵To 4X 10⁶A population of tregs), wherein said population of tregs-containing cells has not been cryopreserved prior to said administration thereof; c) a conventional T cell (Tcon) population, wherein the Tcon is CD3+, wherein the Tcon population comprises less than 1 x 10 per kilogram body weight of the human subject⁷Tcon (preferably, 5 × 10)⁵To 4X 10⁶A Tcon), wherein said Tcon population is cryopreserved prior to said administration thereof; and d) a Graft Versus Host Disease (GVHD) prophylactic agent (preferably tacrolimus or sirolimus), wherein said HSPC population and said population of Treg-containing cells are administered prior to (preferably, 36-60 hours prior to) the administration of said Tcon population, and wherein said human subject does not develop Graft Versus Host Disease (GVHD) within 30 days (preferably, within 1 year) after said administration of said Tcon population.

In some embodiments, the present invention discloses a method of treating a human subject in need of treatment described herein, comprising administering to the human subject: a) a population of Hematopoietic Stem Progenitor Cells (HSPC), wherein the HSPC population comprises more than 1 x 10 per kilogram body weight of the human subject⁵HSPC (preferably, 5X 10)⁵To 2X 10⁷HSPC), wherein the HPSC is CD34⁺(ii) a b) A cell population comprising regulatory T cells (Tregs), wherein the Tregs are CD4⁺CD25⁺CD127^dimOr CD4⁺FOXP3⁺Wherein the population of cells comprising Tregs comprises more than 1 x 10 per kilogram body weight of the human subject⁵Treg (preferably, 5 × 10)⁵To 4X 10⁶A Treg), wherein the population of cells comprising tregs comprises CD45⁺Cells in which more than 50% (preferably, more than 90%) of said CD45⁺The cells are tregs, wherein said population of cells comprising tregs has not been cryopreserved prior to said administration thereof; c) a conventional T cell (Tcon) population, wherein the Tcon is CD3+, wherein the Tcon population comprises less than 1 x 10 per kilogram body weight of the human subject⁷Tcon (preferably, 5 × 10)⁵To 4X 10⁶Tcon); d) A Graft Versus Host Disease (GVHD) prophylactic agent (preferably, tacrolimus or sirolimus), wherein said HSPC population and said population of Treg-containing cells are administered prior to (preferably, 36-60 hours prior to) the administration of said Tcon population, and wherein said human subject does not develop Graft Versus Host Disease (GVHD) within 30 days (preferably, within 1 year) after said administration of said Tcon population.

In some embodiments, the present invention discloses a method of treating a human subject in need of treatment described herein, comprising administering to the human subject: a) a population of Hematopoietic Stem Progenitor Cells (HSPC), wherein the HSPC population comprises more than 1 x 10 per kilogram body weight of the human subject⁵HSPC (preferably, 5X 10)⁵To 2X 10⁷HSPC), wherein the HPSC is CD34⁺(ii) a b) A cell population comprising regulatory T cells (Tregs), wherein the Tregs are CD4⁺CD25⁺CD127^dimOr CD4⁺FOXP3⁺Wherein the population of cells comprising Tregs comprises more than 1 x 10 per kilogram body weight of the human subject⁵Treg (preferably, 5 × 10)⁵To 4X 10⁶A Treg), wherein the population of cells comprising tregs comprises CD45⁺Cells in which more than 50% (preferably, more than 90%) of said CD45⁺The cells are tregs, wherein said population of cells comprising tregs has not been cryopreserved prior to said administration thereof; and c) a conventional T cell (Tcon) population, wherein the Tcon is CD3+, wherein the Tcon population comprises less than 1 x 10 weight per kilogram body weight of the human subject⁷Tcon (preferably, 5 × 10)⁵To 4X 10⁶A Tcon), wherein said Tcon population is cryopreserved prior to said administration thereof, wherein said HSPC population and said Treg-comprising cell population are administered prior to (preferably 36-60 hours prior to) administration of said Tcon population, and wherein said human subject does not develop Graft Versus Host Disease (GVHD) within 30 days (preferably within 1 year) after said administration of said Tcon population.

In some embodiments, the present invention discloses a method of treating a human subject in need of treatment as described hereinComprising administering to the human subject: a) a population of Hematopoietic Stem Progenitor Cells (HSPC), wherein the HSPC population comprises more than 1 x 10 per kilogram body weight of the human subject⁵HSPC (preferably, 5X 10)⁵To 2X 10⁷HSPC), wherein the HPSC is CD34⁺(ii) a b) A cell population comprising regulatory T cells (Tregs), wherein the Tregs are CD4⁺CD25⁺CD127^dimOr CD4⁺FOXP3⁺Wherein the population of cells comprising Tregs comprises more than 1 x 10 per kilogram body weight of the human subject⁵Treg (preferably, 5 × 10)⁵To 4X 10⁶A Treg), wherein the population of cells comprising tregs comprises CD45⁺Cells in which more than 50% (preferably, more than 90%) of said CD45⁺The cells are tregs, wherein said population of cells comprising tregs has not been cryopreserved prior to said administration thereof; c) a conventional T cell (Tcon) population, wherein the Tcon is CD3+, wherein the Tcon population comprises less than 1 x 10 per kilogram body weight of the human subject⁷Tcon (preferably, 5 × 10)⁵To 4X 10⁶A Tcon), wherein said Tcon population is cryopreserved prior to said administration thereof; and d) a Graft Versus Host Disease (GVHD) prophylactic agent (preferably tacrolimus or sirolimus), wherein said HSPC population and said population of Treg-containing cells are administered prior to (preferably 36-60 hours prior to) the administration of said Tcon population.

In some embodiments, the present invention discloses a method of treating a human subject in need of treatment described herein, comprising administering to the human subject: a) a population of Hematopoietic Stem Progenitor Cells (HSPC), wherein the HSPC population comprises more than 1 x 10 per kilogram body weight of the human subject⁵HSPC (preferably, 5X 10)⁵To 2X 10⁷HSPC), wherein the HPSC is CD34⁺(ii) a b) A cell population comprising regulatory T cells (Tregs), wherein the Tregs are CD4⁺CD25⁺CD127^dimOr CD4⁺FOXP3⁺Wherein the population of cells comprising Tregs comprises more than 1 x 10 per kilogram body weight of the human subject⁵Treg (preferably, 5 × 10)⁵To 4X 10⁶A Treg), wherein the population of cells comprising tregs comprises CD45⁺Cells in which more than 50% (preferably, more than 90%) of said CD45⁺The cells are tregs, wherein said population of cells comprising tregs has not been cryopreserved prior to said administration thereof; c) a conventional T cell (Tcon) population, wherein the Tcon is CD3+, wherein the Tcon population comprises less than 1 x 10 per kilogram body weight of the human subject⁷Tcon (preferably, 5 × 10)⁵To 4X 10⁶A Tcon), wherein said Tcon population is cryopreserved prior to said administration thereof; and d) a Graft Versus Host Disease (GVHD) prophylactic agent (preferably tacrolimus or sirolimus), wherein said HSPC population and said population of Treg-containing cells are administered prior to (preferably, 36-60 hours prior to) the administration of said Tcon population, and wherein said human subject does not develop Graft Versus Host Disease (GVHD) within 30 days (preferably, within 1 year) after said administration of said Tcon population.

Examples of the invention

Example 1: clinical trial comprising administration of HSPC and Treg prior to Tcon administration

This example illustrates the utility of the disclosed methods in treating a subject with cancer, including achieving immune reconstitution and GVT effects in the absence of GVHD.

Twelve high-risk malignant disease patients with HLA-matched sibling donors were enrolled in this study. The study was approved by the Stanford institutional review Board (NCT01660607) and FDAIND 14686. Written informed consent was obtained from all patients and donors as specified in the declaration of helsinki.

Design of research

The patient characteristics are shown in figure 1; abbreviations are as follows: TBI, whole body irradiation; cy, cyclophosphamide; VP-16, etoposide; bu, busulfan; AML, acute myeloid leukemia; CR, complete remission; MDS, myelodysplastic syndrome; refractory anemia is caused by the excessive outbreak of RAEB2, type 2; NHL, non-hodgkin lymphoma; ETP-ALL, early precursor T cell acute lymphoblastic leukemia; MRD, minimal residual disease; CML, chronic myelogenous leukemia; MF, myelofibrosis

Following myeloablative conditioning, the subjects were administered the HSPC population and Treg population on day 0, followed by the Tcon population on day 2. The main objectives of this study were: determining the effectiveness, safety and feasibility of the method; determining the maximum tolerated dose of the infused tregs and Tcon; and determining EFS 1 year after HCT. Safety was assessed in terms of any serious adverse event of grade 2 or above resulting from treatment, and specific dose-limiting toxicity was defined as: grade 2 or higher acute GVHD, grade 4 neutropenia lasting 28 days after HCT and grade 3 to 5 cytokine/release syndrome/acute infusion response.

Inclusion criteria included: acute leukemia (primary refractory, CR1, with Minimal Residual Disease (MRD) positive or high-risk features, relapsed after first complete remission), chronic myelogenous leukemia (accelerated or fulminant phase), myelodysplastic syndrome (Int-2 or high-risk), myeloproliferative disorders, non-hodgkin's lymphoma (with low-risk features, not suitable for autologous HCT), age ≤ 60 years, HLA-matched related donors, and prior myeloablative therapy or HCT.

The original clinical protocol is illustrated in figure 2A. Two days prior to Tcon administration, Treg and HSPC donor grafts were administered. After completion of the first cohort recruitment of five patients following the original protocol, GVHD was observed to have occurred. The protocol was modified to take fresh Treg cells instead of frozen Treg cells and introduce targeted doses of either tacrolimus alone (4-6ng/ml) or sirolimus (6-8ng/ml) GVHD prophylactic therapy (figure 2B).

Pretreatment regimen and supportive care

Clinical protocols support the use of different myeloablative conditioning protocols depending on the disease characteristics of the selected subjects. For acute leukemia, high risk CML, myelodysplastic syndrome, and myeloproliferative disorders, pretreatment involves fractionated total body irradiation: fTBI (1320cGy, divided over four days), VP16(60mg/kg, single infusion) and cyclophosphamide (60mg/kg, single infusion); or busulfan (initial 3.6mg/kg, q24, infusion over 4 days, target busulfan levels of 800-900nM) and cyclophosphamide (60mg/kg per infusion, two infusions). For non-Hodgkin's lymphoma, pretreatment included carmustine (300mg/m2), VP16(60mg/kg), and cyclophosphamide (100 mg/kg).

Subjects under the original regimen did not receive GVHD prophylactic therapy, but after the regimen was modified, subjects could receive tacrolimus or sirolimus administration based on preclinical evidence that sirolimus contributes to Treg action. After one subject who received BU/Cy pretreatment developed sinus hepatoobstruction syndrome (SOS), sirolimus GVHD prophylactic therapy was only applicable to subjects who received TBI-based pretreatment.

Infectious disease prevention therapy included acyclovir 400mg (three times daily, oral, for 1 year), single concentration of compound sulfamethoxazole (day 30-60) and ciprofloxacin 500mg (oral, twice daily, day-2 until transplantation). From day +14 to day +100, virus monitoring was performed by quantitative PCR for EBV (once every 2 weeks) and CMV (once a week).

Transplantation engineering

After giving one dose of 10mcg/kg rhG-CSF (Neupogen, Amgen, Qian oak, Calif.) per day and 5 doses, donor cells were harvested from volunteer donors by apheresis at the Stanford Health Care university medical center (SPECTRA; Cobe BCT) using a continuous flow cell Separator (SPECTRA). Two consecutive apheresis collections were performed on

days

4 and 5 and the cell products were mixed. CD34+ cells were collected using either Isolex 300i (Baxter healthcare, dierfield, illinois) or clini macs cell selection system (miltenyi biotec, bergish gladbach, germany). The CD34 reduced (flow-through) fraction was retained and used to isolate donor tregs. Cell selection is performed using clinical grade reagents within a BMT cell therapy facility according to drug production quality regulatory requirements.

CD25+ cells were then selected from CD34 depleted fractions by microbead purification (Miltenyi). Tcon was obtained from the negative fraction and the positive fraction was used for Treg purification. CD4+ CD25+ CD127dim cells were further selected by FACS using a BD infiux cell sorter (BD Biosciences, san jose, california). For CD4 and FoxP3 tablesRepresentative analysis results of cells collected after CD34+ selection, CD25+ cell selection, and CD4+ CD127+ cell sorting are shown in fig. 3. Treg enrichment was shown after depletion of CD34+ cells by immunomagnetic selection (left panel), selection of CD25+ by immunomagnetic selection (middle panel) and sorting of CD4+ CD127lowCD25+ cells by FACS (right panel). A representative chart of an example of a patient is provided herein. From this, it can be seen that tregs of high purity are obtained. These cells are highly inhibitory in Mixed Lymphocyte Reaction (MLR). FIG. 4A illustrates CFSE of T cells (cultured HLA mismatched PBMC with/without Tregs (ratio: 1:1)) by FACS analysis^dimThe proportion of the population in the allogeneic MLR. For MLR, enriched CD4 from donor peripheral blood or apheresis products⁺And CD8⁺Cells (2X 10)⁵MACS microbeads, Miltenyi Biotech) were aliquoted into 96-well flat-bottomed plates as responders. The responders were labeled with CellTrace CFSE on day 0 (Invitrogen, molecular probes) or at 1. mu. Ci/well for the last 16 hours of the 5 day assay according to the manufacturer's instructions³H, thymidine, Perkin-Elmer]The responder is pulsed. The stimulus was a combination of 9 different buffy coats from healthy donors. Using cesium irradiator at 33Gy (¹⁵⁷Cs) is irradiated with a stimulus such that the ratio of stimulus to responder (R: S) is 1:0, 1:1, 1:2 and 1: 4. Tregs were added as inhibitors in the case of responder to inhibitor ratios (R: SUP) of 1:1, 1:0.5, 1:0.25 and 1: 10. To the medium (cRPMI) OKT3(Miltenyi Biotech) was added. Results were read on day 5 by flow cytometry (LSRII BD) or beta read counter (Tomtec, Wallac). Further results are shown in FIGS. 4B-C.

For clinical sorting, the antibodies were repurified by protein a (or G) chromatography and placed in a low pH environment for 30 minutes. The antibody was conjugated to FITC and Alexafluor 647, sterile filtered and stored as a single aliquot.

Dose escalation and implant characterization

Following myeloablative conditioning, the subjects were administered the HSPC population and Treg population on day 0, followed by the Tcon population on day 2.

The study involved a dose escalation protocol as shown in figure 5. Treg dose escalation was determined as the ratio of Treg to Tcon. In the first cohort of three patients, the initial dose cohort target dose was 1 × 10⁶Treg/kg and 3X 10⁶CD3+ Tcon/kg. Since one patient was observed to develop GVHD, three additional subjects were included in the group according to a pre-determined dose escalation protocol, with a Treg: Tcon ratio of 1:1, i.e. 1X 10⁶Treg/kg and 1X 10⁶Tcon/kg. Since no dose limiting toxicity was observed in the subjects otherwise included, the target dose was increased to 3 × 10⁶Treg/kg and 3X 10⁶CD3+ Tcon/kg. After no dose-limiting toxicity was observed in the three additional included patients, the maximum tolerated target dose was determined, which is also the maximum technical dose of tregs that can be collected by two apheresis, and three additional patients were included at this dose level. For many patients, up to 3X 10⁶The dose of Treg/kg is not feasible, but should be administered no matter what cell dose level is achieved.

A schematic of graft generation and administration of fresh tregs following a modified protocol is shown in figure 6. The dosage is 1.0 × 10⁶All subjects who received Treg at this target dose of Treg, but not all received 3.0 x 10⁶Target dose of Treg/kg. FIG. 7 shows the cell dose yield of subjects with a median of 2.5X 10 administered dose⁶Treg/kg (range 2.4-3.0X 10)⁶Treg/kg). Based on FOXP3+ expression, the median Treg recovery obtained with intracellular cytokine staining was 67% (ranging from 50.7-87.8%) and the median Treg purity was 94% (ranging from 91-96%). All other patients had a CD34+ dose, except one patient>2.0×10⁶Single cell/kg, median dose 3.9X 10⁶One cell/kg (range 1.2-15.9X 10)⁶). See figure 8 for dose details.

Cell infusion, transplantation and clinical outcome

The clinical results are summarized in FIG. 9. No infusion reaction was observed. All twelve examplesAll subjects achieved primary complete donor type transplantation, but one received the lowest dose level (1X 10)⁶Treg/kg and 1X 10⁶CD3+ Tcon/kg) in subjects with high risk RAEB-2 MDS. Neutrophils reached 1.0X 10 on the median day of 11 days (range 10-16 days) for all subjects including this example⁹And L. Platelets reach 50X 10 in 16 median days (range 9-25 days)⁹And L. Complete remission was achieved in three of the five patients receiving treatment (duration of remission in 2 of them) prior to protocol modification>For 1 year). After protocol modification (GVHD prophylactic therapy with fresh tregs), complete remission was achieved in five of seven patients (duration of remission in four of them)>1 year), no GVHD was observed.

In patients who initially received Treg: Tcon at a ratio of 1:3, development of grade III acute GVHD was observed. Thus, according to the scheme, at 1 × 10⁶The dose/kg adjusted the ratio of Treg to Tcon to 1:1. In the first three subjects receiving treatment at this dose level, one incidence of acute GVHD (grade III) was observed. Two additional patients were treated at this ratio. Another example of patients developed steroid-reactive grade I acute cutaneous GVHD.

The regimen has been modified to use fresh tregs only and includes low dose single drug GVHD prophylactic therapy with sirolimus as the agent has been shown to protect tregs (figure 2). In clinical situations, if sirolimus administration may increase the risk of hepatic sinus obstruction syndrome, a low dose of tacrolimus is used instead. After protocol modification, no cases of GVHD development were observed in the next seven patients. Although one subject exhibited symptoms of elevated transaminase and received treatment for potential liver GVHD or drug intolerance, rapid regression of symptoms of elevated transaminase was observed following drug changes.

A total of eleven patients with a survival period >6 months can be assessed for chronic GVHD. In the 481 day median follow-up time (ranging from 212-1887 days), two of the ten subjects developed chronic GVHD, both subjects were in the cohort of the original protocol. The manifestations of both subjects with early grade III acute GVHD and chronic GVHD are restricted and steroid-responsive dermatoses. After protocol modification, no patients in the second cohort developed chronic GVHD

One patient who received busulfan and cyclophosphamide pre-treatment and received sirolimus GVHD prophylactic therapy developed mild SOS after protocol modification, and the symptoms resolved after defibrotide treatment and supportive care. Eleven of the twelve patients developed mucositis (4 with grade 1 mucositis; 3 with grade 2; and 4 with grade 3).

Immune reconstitution

To assess the immune reconstitution status of patients enrolled in the study, a comparative standard treatment control cohort was determined, which included five patients who received myeloablative HCT and had no acute or chronic GVHD or no relapse. Using flow cytometry, immune cell subsets in freeze-thawed PBMCs were quantified at standardized time points after HCT (fig. 10).

PBMCs from BMT patients (pre-infused or non-pre-infused tregs) were recovered from liquid nitrogen. PBMC were washed with MACS buffer and either panPBMC plates or T at 4 ℃_regActivated plates were stained for 1 hour. After washing with MACS buffer, the stained cells were fixed with 4% formaldehyde at 4 ℃ overnight. Flow cytometry analysis was performed on LSR II and data analysis was performed using FlowJo software. Activity staining was performed for 5 minutes at room temperature using 0.5. mu.M cisplatin (Fluidigm, cat # 201064). A cell surface antibody premix was prepared and filtered using a 0.2 μm filter. Cells were resuspended in the premix and incubated for 30 minutes. Cells were washed and incubated in a fixative solution (eBiosciences, Cat: 00-5523) prepared according to the manufacturer's recommendations. The cells were fixed at room temperature for 1 hour, washed, and then incubated with a solution of the intracellular antibody mixture in a permeabilization buffer (eBiosciences, Cat: 00-5523) for 1 hour.

Patients in the original protocol cohort exhibited relatively poor T, B, NK and Treg reconstitution capacity compared to patients treated according to the modified protocol or the standard treatment protocol (figure 10). Patients receiving fresh Treg treatment according to the modified protocol typically exhibit comparable immune reconstitution capacity in T, B, NK, DC and monocyte populations compared to the standard treatment control group. The absolute number of iNKT cells was significantly reduced in the first six months in the patient cohort under both the original and modified protocol compared to the control group (two-tailed student T-test, p < 0.01).

Patients receiving fresh Treg treatment according to the modified protocol showed comparable immune reconstitution capacity in terms of Treg proportion and absolute value after HCT compared to standard treated controls without acute or chronic GVHD (two-tailed student T test, p < 0.01). Phenotypic characterization of tregs by flow cytometry revealed no statistical differences in the expression of the surface receptors CTLA-4, PD-1, CD62L, CD45RO or FOXP 3. CyTOF also confirmed this. It was also observed that the intracellular IFN-, TNF-expression appeared not to be increased ex vivo and without stimulation. Fig. 11A illustrates the characteristics of tregs quantified by flow cytometry at day 60 post-HCT (gating on CD4+ CD25+ CD127 dim). Fig. 11B illustrates the characteristics of tregs quantified by cyttof at day 60 post HCT (gating on CD4+ CD25+ CD127 dim).

Treg TCR repertoire analysis

To assess TCR repertoire reconstitution of the Treg compartment post-HCT, CD4+ CD25+ CD127dim tregs taken from patients were purified by FACS around day 90 post-transplantation. For TCR repertoire analysis, tregs were enriched with PBMCs of patients by a two-step isolation procedure: CD25+ cells were isolated from PBMC using CD25MicroBeads II (Miltenyi Biotech) according to the manufacturer's instructions. After staining with anti-CD 3(OKT3, BioLegend), anti-CD 4(OKT4, BioLegend), anti-CD 8(RPA-T8, BioLegend), anti-CD 25(BC96, BioLegend) and anti-CD 127(A019D5, BioLegend) antibodies, CD3+, CD4+, CD8-, CD25+, CD127dim cells were isolated and then stained with anti-CD 3+, CD4+, CD8-, CD25+, CD127dim antibodies

The Plus reagent lysed the cells and placed them at-80 ℃ for storage. 2,700-48,000 Treg cells were isolated.

Taking RNA as a template, and adopting a 5' RACE method and modified gene-specific primers to carry out TCR Rep-Seq in a TCRA or TCRB constant region. The purified 5' RACE PCR products were processed using the KAPA Hyper Prep kit to prepare sequencing libraries. Sequencing was performed by double-ended 250 × 2 cycles using the Illumina MiSeq 500 cycles V2 kit. The double-ended reads of MiSeq reads were submitted to MixCR for TCRA and TCRB rearrangement analysis. The unique CDR3 amino acid sequence (CDR3 clone) was summarized for each sample based on MixCR results. Single copy CDR3 clones were removed. The frequency of clonotypes is calculated by dividing the copy number of a clonotype by the total number of copies of all clonotypes in the sample. Bioinformatics analysis was performed on the data. After testing according to either the standard treatment protocol or the modified clinical trial protocol in which fresh tregs were administered, the results showed no statistical difference in tregs in diversity (fig. 11C).

Claims

1. A method of treating a human subject in need of treatment as described herein, comprising administering to the human subject:

a) a population of Hematopoietic Stem Progenitor Cells (HSPCs);

b) a cell population comprising regulatory T cells (tregs); and

c) a population of conventional T cells (Tcon);

wherein the HSPC population and the Treg-comprising cell population are administered prior to the administration of the Tcon population; and wherein said population of cells comprising tregs has not been cryopreserved prior to said administration thereof.

2. The method according to claim 1, wherein the HSPC is CD34⁺。

3. The method of any one of claims 1-2, wherein the Treg is CD4⁺CD25⁺CD127^dim。

4. The method of any of claims 1-3, wherein the Tcon is CD3⁺。

5. The method of any one of claims 1, 2 and 4, wherein the population of cells comprising Tregs comprises CD45⁺Cells, wherein more than 90% of said CD45⁺The cells are tregs.

6. The method of claim 5, wherein the Treg is CD4⁺CD25⁺CD127^dimOr CD4⁺FOXP3⁺。

7. The method according to any of claims 1-6, wherein the HSPC population comprises more than 1 x 10⁵Individual HSPCs per kilogram body weight of the human subject.

8. The method according to any of claims 1-7, wherein the HSPC population comprises a 5 x 10 population⁵To 2X 10⁷Individual HSPCs per kilogram body weight of the human subject.

9. The method of any one of claims 1-8, wherein the population of cells comprising Tregs comprises more than 1 x 10⁵Individual tregs per kilogram body weight of said human subject.

10. The method of any one of claims 1-9, wherein the population of cells comprising tregs comprises 1 x 10⁵To 1X 10⁷Individual tregs per kilogram body weight of said human subject.

11. The method of any one of claims 1-10, wherein the population of cells comprising tregs comprises 5 x 10⁵To 4X 10⁶Individual tregs per kilogram body weight of said human subject.

12. The method of any of claims 1-11, wherein the Tcon population comprises less than 1 x 10⁷Tcon/kg body weight of the human subject.

13. The method of any one of claims 1-11Method, wherein the Tcon population comprises 1 x 10⁵To 1X 10⁷Tcon/kg body weight of the human subject.

14. The method of any of claims 1-13, wherein the Tcon population comprises 5 x 10⁵To 4X 10⁶Tcon/kg body weight of the human subject.

15. The method of any one of claims 1-14, further comprising administering to the human subject constant natural killer T cells (inkts).

16. The method of claim 15, wherein the iNKT is CD3⁺Vα24Jα18⁺。

17. The method of any one of claims 15-16, wherein the iNKT population comprises more than 5 x 10²Individual inkts per kilogram body weight of the human subject.

18. The method of any one of claims 15-17, wherein the iNKT population comprises 5 x 10²To 1X 10⁷Individual inkts per kilogram body weight of the human subject.

19. The method of any one of claims 1-18, further comprising administering to the human subject a population of memory T cells (Tmem).

20. The method of claim 19, wherein the Tmem is CD3⁺CD45RA^-CD45RO⁺。

21. The method of any one of claims 19-20, wherein the population of Tmem comprises more than 3 x 10⁵One Tmem per kilogram body weight of the human subject.

22. The method of any one of claims 19-21Method, wherein said population of Tmem comprises 3 x 10⁵To 1X 10⁹One Tmem per kilogram body weight of the human subject.

23. The method of any one of claims 1-22, wherein the administering comprises infusing the human subject with the HSPC population, the cell population comprising tregs, and the Tcon population.

24. The method of any one of claims 1-23, wherein the Tcon population is administered at least 12 hours after administration of the HSPC population.

25. The method of any one of claims 1-24, wherein the Tcon population is administered within 24 to 96 hours after administration of the HSPC population.

26. The method of any one of claims 1-25, wherein the Tcon population is administered within 36 to 60 hours after administration of the HSPC population.

27. The method of any one of claims 1-26, wherein the population of Tcon is administered at least 12 hours after the population of cells comprising tregs is administered.

28. The method of any one of claims 1-27, wherein the population of Tcon is administered within 24 to 96 hours after the population of cells comprising tregs is administered.

29. The method of any one of claims 1-28, wherein the Tcon population is administered within 36 to 60 hours after the population of cells comprising tregs is administered.

30. The method of any one of claims 1-29, wherein the human subject does not develop Graft Versus Host Disease (GVHD) within 30 days after the administration of the Tcon population.

31. The method of any one of claims 1-30, wherein the human subject does not develop Graft Versus Host Disease (GVHD) within 100 days after the administration of the Tcon population.

32. The method of any one of claims 1-31, wherein the human subject does not develop Graft Versus Host Disease (GVHD) within 200 days after the administration of the Tcon population.

33. The method of any one of claims 1-32, wherein the human subject has not developed Graft Versus Host Disease (GVHD) within 1 year after the administration of the Tcon population.

34. The method of any one of claims 1-33, wherein the human subject is receiving cancer therapy.

35. The method of any one of claims 1-34, wherein the administration enhances hematopoietic chimerism in the human subject as compared to the human subject's physical condition prior to the administration.

36. The method of any one of claims 1-35, wherein the administration enhances cancer remission in the human subject as compared to the human subject's physical condition prior to the administration.

37. The method according to any one of claims 1-36, wherein the HSPC population is allogeneic to the human subject.

38. The method of any one of claims 1-37, wherein the HSPC population is obtained from a donor HLA-matched to the human subject.

39. The method of any one of claims 1-37, wherein the HSPC population is obtained from a donor HLA-mismatched to the human subject.

40. The method of any one of claims 1-37, wherein the HSPC population is obtained from a donor that is haploid concordant with the human subject.

41. The method of any one of claims 1-40, wherein the population of cells comprising Tregs is allogeneic to the human subject.

42. The method of any one of claims 1-41, wherein the population of cells comprising Tregs is obtained from a donor HLA-matched to the human subject.

43. The method of any one of claims 1-41, wherein the population of cells comprising Tregs is obtained from a donor that is HLA mismatched to the human subject.

44. The method of any one of claims 1-41, wherein the population of cells comprising Tregs is obtained from a donor that is haploid concordant with the human subject.

45. The method of any one of claims 1-44, wherein the Tcon population is allogeneic to the human subject.

46. The method of any one of claims 1-45, wherein the Tcon population is obtained from a donor HLA-matched to the human subject.

47. The method of any one of claims 1-45, wherein the Tcon population is obtained from a donor HLA-mismatched to the human subject.

48. The method of any one of claims 1-45, wherein the Tcon population is obtained from a donor haploid matched to the human subject.

49. The method of any one of claims 1-48, further comprising administering to the human subject a Graft Versus Host Disease (GVHD) prophylactic.

50. The method of claim 49, wherein the GVHD prophylactic agent is tacrolimus or sirolimus.

51. The method of any one of claims 1-50, wherein said population of Tcon is cryopreserved prior to said administration thereof.

52. The method of any one of claims 1-51, wherein the Tcon population is obtained from peripheral blood.

53. A method of treating a human subject in need of treatment as described herein, comprising administering to the human subject:

a) a population of Hematopoietic Stem Progenitor Cells (HSPCs);

b) a cell population comprising regulatory T cells (tregs); and

c) a population of conventional T cells (Tcon);

wherein the population of HSPCs and the population of cells comprising Tregs are administered prior to administration of the population of conventional T cells; and wherein said human subject has not developed Graft Versus Host Disease (GVHD) within 30 days after said administration of said Tcon population.

54. The method according to claim 53, wherein the HSPC is CD34⁺。

55. The method of any one of claims 53-54, wherein the Treg is CD4⁺CD25⁺CD127^dim。

56. The method of any one of claims 53-55, whereinThe Tcon is CD3⁺。

57. The method of any one of claims 53, 54 and 56, wherein the population of cells comprising Tregs comprises CD45⁺Cells, wherein more than 90% of said CD45⁺The cells are tregs.

58. The method of any one of claims 53-54 or 56-57, wherein the Treg is CD4⁺CD25⁺CD127^dimOr CD4⁺FOXP3⁺。

59. The method according to any of claims 53-58, wherein the HSPC population comprises more than 1 x 10⁵Individual HSPCs per kilogram body weight of the human subject.

60. The method according to any one of claims 53-59, wherein the HSPC population comprises 5 x 10⁵To 2X 10⁷Individual HSPCs per kilogram body weight of the human subject.

61. The method of any one of claims 53-60, wherein the population of cells comprising Tregs comprises more than 1 x 10⁵Individual tregs per kilogram body weight of said human subject.

62. The method of any one of claims 53-61, wherein the population of cells comprising Tregs comprises 1 x 10⁵To 1X 10⁷Individual tregs per kilogram body weight of said human subject.

63. The method of any one of claims 53-62, wherein the population of cells comprising Tregs comprises 5 x 10⁵To 4X 10⁶Individual tregs per kilogram body weight of said human subject.

64. The method of any one of claims 53-63, wherein the Tcon population comprises less than 1 x 10⁷Individual Tcon/kg of the human subjectThe body weight of the patient.

65. The method of any one of claims 53-63, wherein the Tcon population comprises 1 x 10⁵To 1X 10⁷Tcon/kg body weight of the human subject.

66. The method of any one of claims 53-65, wherein the Tcon population comprises 5 x 10⁵To 4X 10⁶Tcon/kg body weight of the human subject.

67. The method of any one of claims 53-66, further comprising administering to the human subject constant natural killer T cells (iNKTs).

68. The method of claim 67, wherein the iNKT is CD3⁺Vα24Jα18⁺。

69. The method of any one of claims 67-68, wherein the iNKT population comprises more than 5 x 10²Individual inkts per kilogram body weight of the human subject.

70. The method of any one of claims 67-69, wherein the iNKT population comprises 5 x 10²To 1X 10⁷Individual inkts per kilogram body weight of the human subject.

71. The method of any one of claims 53-70, further comprising administering to the human subject a population of memory T cells (Tmem).

72. The method of claim 71, wherein the Tmem is CD3⁺CD45RA^-CD45RO⁺。

73. The method of any one of claims 71-72, wherein the Tmem population comprises more than 3 x 10⁵One Tmem/kg of said human subjectThe body weight of the patient.

74. The method of any one of claims 71-72, wherein the Tmem population comprises 3 x 10⁵To 1X 10⁹One Tmem per kilogram body weight of the human subject.

75. The method of any one of claims 53-74, wherein the administering comprises infusing the HSPC population, the cell population comprising Tregs and the Tcon population into the human subject.

76. The method of any one of claims 53-75, wherein the Tcon population is administered at least 12 hours after administration of the HSPC population.

77. The method of any one of claims 53-76, wherein the Tcon population is administered within 24 to 96 hours after administration of the HSPC population.

78. The method of any one of claims 53-77, wherein the Tcon population is administered within 36 to 60 hours after administration of the HSPC population.

79. The method of any one of claims 53-78, wherein the Tcon population is administered at least 12 hours after the Treg-comprising cell population is administered.

80. The method of any one of claims 53-79, wherein the Tcon population is administered within 24 to 96 hours after the Treg-comprising cell population is administered.

81. The method of any one of claims 53-80, wherein the Tcon population is administered within 36 to 60 hours after the Treg-comprising cell population is administered.

82. The method of any one of claims 53-81, wherein the human subject does not develop Graft Versus Host Disease (GVHD) within 100 days after the administration of the Tcon population.

83. The method of any one of claims 53-82, wherein the human subject does not develop Graft Versus Host Disease (GVHD) within 200 days after the administration of the Tcon population.

84. The method of any one of claims 53-83, wherein the human subject does not develop Graft Versus Host Disease (GVHD) within 1 year after the administration of the Tcon population.

85. The method of any one of claims 53-84, wherein the human subject is receiving treatment for cancer.

86. The method of any one of claims 53-85, wherein the administration enhances hematopoietic chimerism in the human subject as compared to the human subject's physical condition prior to the administration.

87. The method of any one of claims 53-86, wherein said administering enhances cancer remission in said human subject as compared to the physical condition of said human subject prior to said administering.

88. The method according to any one of claims 53-87, wherein the HSPC population is allogeneic to the human subject.

89. The method according to any one of claims 53-88, wherein the HSPC population is obtained from a donor HLA-matched to the human subject.

90. The method according to any one of claims 53-88, wherein the HSPC population is obtained from a donor HLA-mismatched to the human subject.

91. The method of any one of claims 53-88, wherein the HSPC population is obtained from a donor haploid compatible with the human subject.

92. The method of any one of claims 53-91, wherein the population of cells comprising Tregs is allogeneic to the human subject.

93. The method of any one of claims 53-92, wherein the population of cells comprising Tregs is obtained from a donor HLA-matched to the human subject.

94. The method of any one of claims 53-92, wherein the population of cells comprising Tregs is obtained from a donor that is HLA mismatched to the human subject.

95. The method of any one of claims 53-92, wherein the population of cells comprising Tregs is obtained from a donor that is haploid concordant with the human subject.

96. The method of any one of claims 53-95, wherein the population of Tcon is allogeneic to the human subject.

97. The method of any one of claims 53-96, wherein the Tcon population is obtained from a donor HLA-matched to the human subject.

98. The method of any one of claims 53-96, wherein the Tcon population is obtained from a donor HLA-mismatched to the human subject.

99. The method of any of claims 53-96, wherein the Tcon population is obtained from a donor haploid matched to the human subject.

100. The method of any one of claims 53-99, further comprising administering to the human subject a Graft Versus Host Disease (GVHD) prophylactic.

101. The method of claim 100, wherein the GVHD prophylactic agent is tacrolimus or sirolimus.

102. The method of any one of claims 53-101, wherein said population of cells comprising Tregs has not been cryopreserved prior to said administration thereof.

103. The method of any one of claims 53-102, wherein said population of Tcon is cryopreserved prior to said administration thereof.

104. The method of any one of claims 53-103, wherein the Tcon population is obtained from peripheral blood.

105. A method of treating a human subject in need of treatment as described herein, comprising administering to the human subject:

a) a population of Hematopoietic Stem Progenitor Cells (HSPCs);

b) a cell population comprising regulatory T cells (tregs); and

c) a population of conventional T cells (Tcon);

wherein the HSPC population and the Treg-comprising cell population are administered prior to the administration of the Tcon population; wherein the population of cells comprising Tregs comprises CD45⁺Cells, wherein more than 90% of said CD45⁺The cells are tregs.

106. The method according to claim 105, wherein the HSPC is CD34⁺。

107. The method as set forth in any one of claims 105-106, wherein the Treg is CD4⁺CD25⁺CD127^dimOr CD4⁺FOXP3⁺。

108. The method as claimed in any one of claims 105-107, wherein the Tcon is CD3⁺。

109. The method according to any one of claims 105-108, wherein the HSPC population comprises more than 1 x 10⁵Individual HSPCs per kilogram body weight of the human subject.

110. The method according to any one of claims 105-109, wherein the HSPC population comprises a 5 x 10 population⁵To 2X 10⁷Individual HSPCs per kilogram body weight of the human subject.

111. The method of any one of claims 105-110, wherein the population of cells comprising tregs comprises more than 1 x 10⁵Individual tregs per kilogram body weight of said human subject.

112. The method of any one of claims 105-111, wherein the population of cells comprising tregs comprises 1 x 10⁵To 1X 10⁷Individual tregs per kilogram body weight of said human subject.

113. The method of any one of claims 105-112, wherein the population of cells comprising tregs comprises 5 x 10⁵To 4X 10⁶Individual tregs per kilogram body weight of said human subject.

114. The method as defined in any one of claims 105-113, wherein the Tcon population comprises less than 1 x 10⁷Tcon/kg body weight of the human subject.

115. The method as defined in any one of claims 105-113, wherein the Tcon population comprises 1 x 10⁵To 1X 10⁷Tcon/kg body weight of the human subject.

116. The method as defined in any one of claims 105-115, wherein the Tcon population comprises 5 x 10⁵To 4X 10⁶Tcon/kg body weight of the human subject.

117. The method of any one of claims 105-116, further comprising administering to the human subject constant natural killer T cells (inkts).

118. The method of claim 117, wherein the iNKT is CD3⁺Vα24Jα18⁺。

119. The method of any one of claims 117-118, wherein the iNKT population comprises more than 5 x 10²Individual inkts per kilogram body weight of the human subject.

120. The method of any one of claims 117-118, wherein the iNKT population comprises 5 x 10²To 1X 10⁷Individual inkts per kilogram body weight of the human subject.

121. The method of any one of claims 105-121, further comprising administering a population of memory T cells (Tmem) to the human subject.

122. The method of claim 121, wherein the Tmem is CD3⁺CD45RA^-CD45RO⁺。

123. The method of any one of claims 121-122 wherein the population of Tmem comprises more than 3 x 10⁵One Tmem per kilogram body weight of the human subject.

124. The method of any one of claims 121-122, wherein the population of Tmem comprises 3 x 10⁵To 1X 10⁹One Tmem per kilogram body weight of the human subject.

125. The method of any one of claims 105-124, wherein the administering comprises infusing the human subject with the HSPC population, the cell population comprising tregs, and the Tcon population.

126. The method according to any one of claims 105-125, wherein the Tcon population is administered at least 12 hours after administration of the HSPC population.

127. The method according to any one of claims 105-126, wherein the Tcon population is administered within 24 to 96 hours after administration of the HSPC population.

128. The method according to any one of claims 105-127, wherein the Tcon population is administered within 36 to 60 hours after administration of the HSPC population.

129. The method of any one of claims 105-128, wherein the Tcon population is administered at least 12 hours after the population of cells comprising tregs is administered.

130. The method of any one of claims 105-129, wherein the Tcon population is administered within 24 to 96 hours after the Treg-comprising cell population is administered.

131. The method of any one of claims 105-130, wherein the Tcon population is administered within 36 to 60 hours after the Treg-comprising cell population is administered.

132. The method of any one of claims 105-131, wherein the human subject has not developed Graft Versus Host Disease (GVHD) within 30 days after the administration of the Tcon population.

133. The method of any one of claims 105-132, wherein the human subject has not developed Graft Versus Host Disease (GVHD) within 100 days after the administration of the Tcon population.

134. The method of any one of claims 105-133, wherein the human subject has not developed Graft Versus Host Disease (GVHD) within 200 days after the administration of the Tcon population.

135. The method of any one of claims 105-134, wherein the human subject has not developed Graft Versus Host Disease (GVHD) within 1 year after the administration of the Tcon population.

136. The method of any one of claims 105-135, wherein the human subject is undergoing treatment for cancer.

137. The method of any one of claims 105-136, wherein the administration enhances hematopoietic chimerism in the human subject as compared to the physical condition of the human subject prior to the administration.

138. The method of any one of claims 105-137, wherein the administration enhances cancer remission in the human subject as compared to the physical condition of the human subject prior to the administration.

139. The method of any one of claims 105-138, wherein the HSPC population is allogeneic to the human subject.

140. The method according to any one of claims 105-139, wherein the HSPC population is obtained from a donor HLA-matched to the human subject.

141. The method according to any one of claims 105-139, wherein the HSPC population is obtained from a donor that is HLA-mismatched to the human subject.

142. The method of any one of claims 105-139, wherein the HSPC population is obtained from a donor that is haploid concordant with the human subject.

143. The method of any one of claims 105-142, wherein the population of cells comprising tregs is allogeneic to the human subject.

144. The method of any one of claims 105-143, wherein the population of cells comprising tregs is obtained from an HLA-matched donor to the human subject.

145. The method of any one of claims 105-143, wherein the population of cells comprising tregs is obtained from a donor that is HLA-mismatched to the human subject.

146. The method of any one of claims 105-143, wherein the population of cells comprising tregs is obtained from a donor that is haploid concordant with the human subject.

147. The method of any one of claims 105-146, wherein the Tcon population is allogeneic to the human subject.

148. The method as defined in any one of claims 105-147, wherein the Tcon population is obtained from a donor HLA-matched to the human subject.

149. The method as defined in any one of claims 105-147, wherein the Tcon population is obtained from a donor HLA-mismatched to the human subject.

150. The method of any one of claims 105-147, wherein the Tcon population is obtained from a donor that is haploid in relation to the human subject.

151. The method of any one of claims 105-150, further comprising administering to the human subject a Graft Versus Host Disease (GVHD) prophylactic agent.

152. The method of claim 151, wherein the GVHD prophylactic agent is tacrolimus or sirolimus.

153. The method of any one of claims 105-152, wherein the population of cells comprising tregs is not cryopreserved prior to the administration thereof.

154. The method of any one of claims 105-153, wherein the Tcon population is cryopreserved prior to the administration thereof.

155. The method of any one of claims 105-154, wherein the Tcon population is obtained from peripheral blood.